The submandibular rat 1 protein (SMR1) is selectively processed at pairs of basic amino acid residues in a tissue- and sex-specific manner. We have mapped peripheral targets for the final secretory maturation product of SMR1, the pentapeptide QHNPR, by examining in vivo the tissue distribution of the radiolabeled peptide using β-radio imager whole body autoradiography. The characteristics of tissue uptake allowed specific binding sites at physiological peptide concentrations to be identified within the renal outer medulla, bone and dental tissue, glandular gastric mucosa, and pancreatic lobules. Direct evidence that pentapeptide binding sites are localized in selective portions of the male rat nephron, within the S3, S2, and S1 segments of the proximal tubules, was obtained. In bone tissue the pentapeptide exclusively accumulates within the trabecular bone remodeling unit, and in dental tissue it concentrates within the tubules of the dentinal rat incisor. In relation to male rat-specific behavioral characteristics, our data suggest that the circulating androgen-regulated SMR1-derived pentapeptide is primarily involved in the modulation of mineral balance between at least four systems: kidney, bone, tooth, and circulation.
- submandibular rat 1 protein-derived peptides
- whole body autoradiography
- β-radio imager quantification
the three major salivary glands, parotid, sublingual, and submandibular glands (SMG), and their secretions are intimately involved in many activities that influence the overall nutritional well-being of mammals.
In addition to the products involved locally, the salivary glands of certain rodents, under multifactorial neuroendocrine control, also produce and secrete into the bloodstream biologically active polypeptides such as growth factors, hormones, or processing enzymes (1, 2, 7). Convincing evidence from experiments of physiological significance suggests that rodent salivary glands have a systemic role as a source of blood circulating factors. Furthermore, the fact that environmental stress conditions, such as aggressive behavior between males, lead to the systemic secretion of some of its peptidic products has suggested that the SMG participates in the control of some species-specific behavioral characteristics of male rodents (4, 14, 18,19, 29).
We have previously characterized a new rat submandibular gland protein, named submandibular rat 1 protein (SMR1), that has the structure of a prohormone and whose synthesis is upregulated by androgen (22). The gene encoding SMR1 belongs to a new multigene family, the Variable Coding Sequence family, which has been localized to chromosome 14, bands p21-p22 (9, 20). The gene has a similar organization to a number of hormone precursor genes. SMR1 mRNA is expressed in a highly tissue-, age-, and sex-specific manner in the acinar cells of the male rat SMG and in the prostate (21).
We have demonstrated that, in vivo, SMR1 is selectively processed at pairs of basic amino acid sites in a tissue- and sex-specific manner to give rise to mature peptide products, in a manner similar to the maturation pathway of peptide-hormone precursors (24). This selective proteolytic fragmentation is critical for the generation and the regulation of biologically active peptides (16). The biosynthesis of the peptides generated from SMR1 by cleavage at pairs of arginine residues (e.g., the undecapeptide VRGPRRQHNPR, the hexapeptide RQHNPR, and the pentapeptide QHNPR) is subject to distinct regulatory pathways depending on 1) the organ, SMG and prostate; 2) the developmental stage, from 6 wk postnatal;3) the sex, predominantly in the male; and 4) gonad hormones, the androgens. Furthermore, in vivo, the mature peptides, which accumulate in the male rat SMG, are exported into the extracellular space in response to a specific external stimulus and in this way are transported within the salivary and blood fluids (24). The fact that these peptides are mainly produced in postpubescent male rats and are secreted into the saliva and blood under stimulated conditions led us to postulate that they have a local and systemic physiological role in mediating some male-specific behavioral characteristics. Thus the ability to determine in the male rat the precise topographical distribution of target organs for SMR1-derived peptides will undoubtedly help in understanding the role of these peptides in local and peripheral systems.
In the present study, we have mapped potential peripheral targets for the final secretory maturation product of SMR1, the pentapeptide, by examining in vivo the gross tissue distribution of the radiolabeled peptide with whole body autoradiography (WBA) (30). This method provides a visual image of the entire animal, therefore rendering it possible to examine simultaneously the uptake of the pentapeptide in a large number of tissues and compartments. Furthermore, this approach is considerably more attractive than an in vitro method, because labeling in living tissue minimizes the possibility of misinterpretation due to nonspecific distribution, to receptor site degradation, and to non-receptor site-bound peptide eliminated through the bloodstream (17). For these reasons, the time-related tissue uptake for the pentapeptide was assessed in vivo. To eliminate the possibility of competition for the available binding sites from endogenous peptide, labeling was achieved by intravenously infusing tritiated pentapeptide in 5-wk-old pubescent rats. Indeed, we have previously shown that the acute generation of the SMR1 maturation peptides appears in the male rat SMG from 6 wk postnatally (24). In addition to film autoradiography, the mapping and the time course of the tritiated peptide uptake by the living organs were recorded by the recently developed β-radio imager, which offers the unique capability of detecting and quantifying the β-particles emitted from tritiated ligand bound to flat surfaces such as whole body sections (28). We also analyzed the chromatographic characteristics of the labeled compound associated with the target organ.
Biological receptor sites have two essential characteristics that allow specific binding to be identified in vivo: they have a high affinity for the ligand and the binding is saturable (31, 32). Therefore, displacement experiments using parallel systemic administration of an excess of the corresponding unlabeled peptide were performed. Furthermore, the specific cellular localization of labeled pentapeptide to target tissues was identified by section microscopic autoradiography.
Finally, to appreciate the relevance of the pentapeptide uptake under physiological conditions, we have determined the endogenous bloodstream secretion pattern of the peptides related to SMR1 in conscious adult male rats. It is important to stress that all the pharmacokinetic and cellular-site distribution studies were realized using physiological concentrations of labeled pentapeptide.
MATERIALS AND METHODS
The peptides corresponding to the sequences (Glp1)-His-Asn-Pro-Arg, (Gln1)-His-Asn-Pro-Arg, and (Gln1)-His-Asn-(Δ3,4-Pro)-Arg were synthesized by the Laboratoire de Chimie Organique, Institut Pasteur, Paris, France. The labeled compound (Glp1/Gln1)-His-Asn-[3,43H]Pro-Arg was synthesized by Dr. R. Genet, Departement d’ingénierie et d’étude des protéines, Commissariat àl’Énergie Atomique/Saclay, Gif-sur-Yvette, France. The reverse phase (RP)-C18high-performance liquid chromatography (HPLC)-purified product (>98% purity) with a specific radioactivity estimated as 2.22 TBq/mmol was stored at −80°C in 10% methanol-0.1% trifluoroacetic acid (1.11 GBq/ml). The purity of the tritiated pentapeptide was systematically assessed before use by reverse-phase C18 and cation-exchange FPLC chromatographies according to the methods previously described (24).
Wistar male and female rats (4 wk old), purchased from Iffa-Credo (France), were kept 2–4 animals/cage under controlled lighting and temperature with free access to food and water until use 5–7 days later for the study of pentapeptide receptor sites distribution and 5–6 wk later for the study of endogenous pentapeptide secretion. They were gently handled daily by the operator throughout this period. All experiments were performed between 1000 and 1400.
Whole Body Macroautoradiographic Visualization
The target tissues for the final secretory maturation product of SMR1, the pentapeptide, were examined in vivo by the WBA procedure according to the method of Ullberg and Larsson (30).
The 5-wk-old, 100-g-body wt rats were killed while under halothane anesthesia at 90 s or 3, 60, or 240 min after intravenous injection of 3 nmol (6.7 MBq or 2 μg) of tritiated pentapeptide. At the selected time, the restrained animal was immediately immersed in a −80°C mixture of dry ice and isopentane to prevent artifactual tracer redistribution. After 48 h in self-sealing plastic stored at −30°C, the animal was then blocked in mounting medium. Whole body sagittal sections (20 μm) of the frozen rat were made at −30°C with a cryostat (whole body slicing microtome Leitz 400 with a chest mobile freezer Leitz OM, Leica). Sections adhering to Scotch tape were left in a freezer, temperature −30°C, for 4 days to ensure complete drying. The tapes were placed in a film cassette with 3H Hyperfilm (Amersham, France) at −20°C. After 2 wk, the films were developed in Kodak D19 developer and fixed in Kodak fixer.
Tissue Preparation for Sectioning and Light Microautoradiographic Visualization
The cellular site localization for physiological concentrations of labeled pentapeptide was investigated by paraffin- or resin-section microscopic autoradiography procedure. Such a level of resolution can only be achieved by directly coating the in vivo radiolabeled organ sections with radiosensitive liquid nuclear emulsion, provided that the bound radioactive peptide is securely cross-linked to the binding sites with divalent aldehydes (17).
Sixty minutes after [3H]pentapeptide (555 kBq, 160 ng or 250 pmol) intravenous administration, the anesthetized male rats were perfused via the jugular veins with ice-cold Krebs-Ringer bicarbonate glucose buffer and then 0.5% paraformaldehyde-0.5% glutaraldehyde fixative phosphate-buffered saline (PBS) buffer (plus 1.6% glucose, 0.002% CaCl2, and 1% dimethyl sulfoxide) (50 ml/5 min for each). Tissues were rapidly removed and harvested for paraffin sectioning (Paraplast plus, Sherwood Medical, OSI) or for resin sectioning (histo-resin, Leica).
All sections were cut at 5 μm (Reichert Jung for paraffin and RM2155 Leica for resin sections) and mounted on Superfrost/Plus glass slides (non-gelatin coated). Paraffin was removed with xylene, and sections were carried through a descending and then an ascending ethanol series (from 100 to 50%, vol/vol, and the reverse). Dried sections were then processed for light microscope autoradiography by attaching nuclear emulsion-coated (Kodak, NTB2, diluted 1:1 with distilled water) coverslips to them. After air drying for 2 h at room temperature, the autoradiographs were exposed for 6–10 wk in light-tight boxes at 4°C. Radiosensitive coverslips were developed in Kodak D19 (3 min) developer and fixed in Kodak fixer (3 min). Sections were counterstained with Harris’ hematoxylin (Prolabo) and toluidine blue (Sigma), dehydrated, and mounted in Eukitt medium. The tissue and overlying silver grains were viewed and photographed with a Leica photomicroscope equipped with bright-field optics (DMRD Leitz, Leica).
Mapping and Quantification of [3H]pentapeptide Uptake by Living Tissues
The quantitative determination of the radioactivity in various organ sections was carried out by using a gaseous detector ofβ-particles that has recently been developed (28). Data from whole body sections or individual organ sections placed in the gas chamber detector were collected for 8–14 h or 50 h, respectively. The number of counts per pixel was recorded in the β-imager 2200 (Biospace, France) with a surface detector of 20 × 20 cm2, followed by computer-assisted image analysis with the β-vision program using a Hewlett-Packard Vectra computer. The tritium activity was determined as counts per square millimeter. The linearity of this method of detection allowed measurement of the nonspecific binding of [3H]pentapeptide defined within each of the sections. This was measured in various structures or compartments with apparent nonbinding sites for the peptide (background tissues, e.g., muscle and cardiac blood except for brain and spinal cord) or in the same labeled structure in displacement experiments.
In the case of radioactive quantification of organ sections or acid extracts, with the use of a liquid scintillation counter (MR300 Kontron), the tritium activity was determined as counts per minute per milligram protein. Protein concentration was determined by the Bradford method (8).
Chromatographic Characterization of Radioactive Peptide Uptake by Tissues
The chromatographic characteristics of the labeled compound associated, in vivo, to target organs were assessed by HPLC.
At selected time points (2 and 180 min) after administration of tritiated pentapeptide, the anesthetized rats were killed (cardiac blood puncture) and the tissues were quickly removed and placed on ice. The tissues were immediately homogenized at 4°C in 5–10 volumes of 0.1 M chlorhydric acid with a Potter homogenizer (demineralization of bone tissue was realized after 24–48 h at 4°C in guanidium hydrochloride and EDTA, 4 and 0.25 M final concentration, respectively). Homogenates were centrifuged for 30 min at 4°C and 15,000 g. To determine total radioactivity, aliquots of the supernatant solution were added to 50 volumes of Biofluor (New England Nuclear-Dupont de Nemours) and counted in a liquid scintillation counter.
To determine the radioactivity in the pentapeptide fraction, aliquots of the tissue extracts were submitted to a methanol extraction procedure (24) after neutralization with tris(hydroxymethyl)aminomethane ⋅ HCl, pH 8.5, containing 16 mM diethylenetriaminepentaacetic acid (DTPA; Sigma). The methanol phase was removed from the supernatant by partial evaporation, followed by lyophilization, and the reconstituted aqueous phase containing 16 mM DTPA was applied to an octadecylsilane aqueous (ODS AQ) column (HPLC RP C18 chromatography) (AIT). Elution with a linear gradient of 0.1% trifluoroacetic acid (TFA) in water-0.1% TFA in acetonitrile (Merck) from 100:0 (vol/vol) to 50:50 (vol/vol) was performed for 30 min at a flow rate of 1 ml/min. Fractions were collected every 60 s and analyzed for radioactivity using a liquid scintillation counter.
Pentapeptide Bloodstream Secretion and Pharmacokinetics In Vivo
Pentapeptide systemic secretion under basal or ether stress- and adrenergic agent-induced SMG secretion of conscious male rats. The in vivo endogenous bloodstream secretion of peptides related to the SMR1 precursor, in particular the pentapeptide, was investigated in conscious adult male rats.
Rats at 9–10 wk of age were placed in a jar for a 2-min exposure to ether fumes or were placed for 20–40 min in individual metabolic cages after intraperitoneal administration of adrenergic secretagogue agents (phenylephrine, 4 mg/kg, plus isoproterenol, 1 mg/kg) or vehicle (PBS-Dulbecco’s medium). At selected times, the rats were anesthetized with pentobarbital sodium (45 mg/kg). Blood samples were taken by cardiac puncture and collected into previously cooled tubes containing a mixture of peptidase inhibitors [1 mM EDTA, 1,000 U/ml aprotinin, 130 μM bestatin, 1 μM leupeptin, 0.4 mM Pefabloc (Boehringer Mannheim, Indianapolis, IN), 1 μM pepstatin]. They were immediately centrifuged for 15 min at 4,000g and 4°C. Plasma fractions were then submitted to the Porapak Q column extraction procedure (23) and tested for the pentapeptide content before and after HPLC separations.
Briefly, acidified plasma samples (HCl, 0.1 N final concentration) were applied to Porapak Q beads (Waters, France) packed in a 0.5 × 2 cm column. After washing with 0.1% TFA in water, the peptides were eluted with 0.1% TFA in 50% methanol (recovery of the marker pentapeptide added was 93 ± 6%,n = 5). Each of the extracted samples was then analyzed by using a HPLC system (Spectra-Physics SP8000) connected to a HEMA-IEC BIO-1000 carboxymethyl column (Alltech). Cation-exchange chromatography was performed with a one-step, 30-min linear gradient of 1–1,000 mM ammonium acetate, pH 4.6, at a 1 ml/min flow rate. Fractions of 1 ml were collected and tested after lyophilization for their pentapeptide content. The radioimmunoassay procedure and characteristics for pentapeptide measurements have been described previously (24).
Pentapeptide metabolic and pharmacokinetic studies. The in vivo time course of distribution, metabolism, and elimination of the pentapeptide was investigated in anesthetized adult male rats using physiological concentrations of circulating tritiated pentapeptide.
Anesthetized male rats were given a bolus intravenous injection of 170 pmol or 110 ng tritiated pentapeptide diluted in 100 μl PBS-Dulbecco’s medium. Blood was collected in tubes containing a mixture of peptidase inhibitors described above via a Silastic catheter implanted into the external jugular vein. Blood was withdrawn just before and 2, 4, 6, 8, 10, 20, 30, 45, 60, 90, and 120 min after peptide administration. Two milliliters of blood per rat were taken in total, and the entire urinary bladder content was collected at the last selected point.
The biological samples were submitted to centrifugation and Porapak Q extraction conditions, as described above. Chromatography using the HPLC procedure described above and in Ref. 24 was then applied to each extracted sample. The ODS AQ RP-C18 was used for identification of amino-terminal metabolites, and PepRPC high-resolution C18 (Pharmacia, France) was used for identification of carboxy-terminal metabolites. The radioactivity content of samples before and after chromatographic separations was determined using a beta counter (Kontron, MR300).
Distribution of Target Organs for3H-Labeled SMR1-Derived Pentapeptide in Male and Female Rats Examined by WBA
WBA has considerable application in studies of receptor binding and the analysis of the biological fate of proteins and peptides. We applied this approach to the examination of the potential target tissues of the SMG-derived SMR1-pentapeptide. The mapping and time course of tissue distribution of radioactive pentapeptide was investigated after [3H]pentapeptide was systemically injected into 5-wk-old male and female rats. To generate autoradiographic images of sufficient density in a reasonable exposure time and to appreciate the specific uptake compared with nonspecific labeling, a quantity of 6.7 MBq isotope was employed in these experiments. Although we have used tritiated peptide of high specific activity, the dose of peptide infused into the circulation resulted in amounts of blood pentapeptide ∼10-fold its physiological level (see below).
The gross anatomic distribution of [3H]pentapeptide uptake at 60 min postdose on whole male rat body sagittal sections is shown in Figs. 1 and2, A,B, andE. As illustrated in representative autoradiograms (Fig. 1, A andB), 60 min after a bolus intravenous injection of radioactive pentapeptide, a dense and distinct accumulation of silver grains was apparent in the kidney and all bone tissue as well as dental tissue, the glandular mucosa of the stomach, pancreatic lobules, and the SMG. At this selected time, moderate levels of labeling were seen in the liver, spleen, thymus, and intestinal wall. Neither the brain nor spinal cord accumulated radioactive pentapeptide, demonstrating that a stringent blood-brain barrier limited the peptide uptake in vivo.
Assessment of tissue peptide targeting was accomplished by β-radio imager quantification (Fig. 2). The number of β-particles emitted per unit area was collected directly from whole body sections. The linearity and selectivity of this method of detection allowed a relevant measurement of labeled structures compared with blackening defined within a selected anatomic area of the same section (here, blood in the cardiac space) (28).
In the kidney, pancreas, bone, incisor, SMG, and glandular gastric mucosa of males, the concentration of radioactivity at 60 min postdose, in counts per square millimeter per 8 h, was markedly higher, 4–10 times, than that of the blood in the heart. Much more radioactivity was present in these tissue spaces than is predicted by the same blood anatomic space (Fig. 2 E). This overestimation of the anatomic plasma and interstitial spaces may reflect the presence of peptide receptor site binding within these tissues (32). In the liver, thymus, intestinal wall, and spleen, the relative tissue accumulation was equal or less than twofold compared with the blackening. In all the other tissues visualized (muscle, gonad, cartilage, nonglandular gastric mucosa, brain), the level of radioactivity was equal to or less than in that blood area, reflecting restriction of the peptide solely to the anatomic plasma and interstitial spaces of these organs (Fig.2 E).
The dynamic profile of the [3H]pentapeptide uptake in males revealed that the radioactivity is rapidly distributed and differentially accumulated as early as 90 s and 3 min, in the kidney, pancreas, dental tissue, and gastric glandular wall, with a tissue-to-blood ratio of close to 2. Label persisted within these tissues for >240 min. More precisely, within the kidney, labeling was predominantly in the outer medullary area, over the time course studied from 90 s to 240 min postinjection (Fig.1 A, 60 min postinjection). Within the bone, labeling was predominantly visualized as early as 90 s and 3 min on the periosteal bone surfaces (periosteum). Within the tooth, labeling also rapidly and stably accumulated in the inner bulk of the incisor root as well as the alveolar bone. By contrast, in the blood and blood-rich organs such as the lung and muscles, radioactivity was widely distributed at 90 s and 3 min but was completely excluded by 60 min. No radioactivity was found in the intestinal content as late as 240 min postinjection, whereas the bladder had radioactivity, indicating that some of the pentapeptide and/or metabolites had already been excreted into the urine. This is consistent with the high labeling found in the renal pelvis and no labeling in the liver 90 s and 3 min after systemic injection. This result may be explained by a more efficient elimination of bloodstream pentapeptide through glomerular filtration than hepatic clearance and biliary excretion.
The in vivo tissue distribution analysis of the radiolabeled peptide by β-radio imager WBA of the 5-wk-old female rat showed the same selectivity of pentapeptide tissue targeting as in the male (Fig. 2,C andD). However, in the male, the rate of radioactivity differentially accumulated 60 min postinjection was found to be relatively well balanced from one target tissue to another (from 6% of the total count for SMG to 10% for bone femur, and at a maximum of 16% for the kidney) (Fig. 2,A, B, and E). In contrast, in the female 60 min postinjection, the radioactivity was mainly taken up by the renal outer medulla (41% of the total count) and by the SMG (6% of the total count). It was found to be poorly accumulated in the other tissues, two- to threefold the blackening in bone and dental tissues as well as pancreatic lobules and gastric mucosa, which represents marginal labeling compared with males.
To determine whether the radioactive species detected is actually the compound administered, acid homogenates from target organs were analyzed after methanol extraction and RP chromatography, 2–20 min and 180–200 min after systemic injection of 2 μg or 3 nmol [3H]pentapeptide. A good correlation was obtained between the β-radio imager and liquid scintillation counting values for each organ, in counts per square millimeter and counts per minute per gram tissue weight, respectively. After 5- to 20-min survival times, almost all the radioactivity uptake by tissues was extracted from the acid homogenates by the organic solvent used: 72 ± 4%. On the other hand, 180 min after injection of [3H]pentapeptide, radioactive peptide fraction was picked up only when the methanol extraction was performed from tissue homogenates to which DTPA, a strong metal chelating agent, was added: 53 ± 9%. Moreover, only under this extraction condition did RP-HPLC chromatography reveal that the radioactivity was predominantly recovered in the peak corresponding to free pentapeptide: 52 ± 8%. The rest of the radioactive extract represented metabolites or undissociated peptide complexes: 8 ± 6 and 29 ± 14%, respectively.
Taken together, these data provide strong evidence of selective, rapid, and stable uptake of the pentapeptide (at least) in the male rat by the outer medulla of the kidney, by the pancreatic lobules, by the glandular mucosa of stomach, and by both bone and dental tissues.
SMR1-Derived Pentapeptide Bloodstream Secretion Pattern in the Adult Male Rat
To appreciate the relevance of the pentapeptide uptake under physiological conditions, the endogenous bloodstream concentration of peptides related to the SMR1 precursor protein, in particular of the pentapeptide, was investigated in conscious adult male rats in response to pharmacological and acute stress stimuli. Blood sampling and extraction described in materials and methods were carried out in the presence of a mixture of peptidase inhibitors plus EDTA, and, under these conditions, the basal plasma pentapeptide-immunoreactive level of 10-wk-old male rats was 1.9 ± 0.2 ng/ml, n = 5. In anesthetized rats, the time course response to adrenergic secretagogue agents of SMR1-derived peptide blood secretion had previously revealed that the peptides showed maximal circulating levels within 10–30 min after peritoneal administration of epinephrine (24). In conscious male rats, 20–40 min after injection of 4 mg/kg phenylephrine and 1 mg/kg isoproterenol, the plasma peptide-immunoreactive response was found to be 12.5 ± 5.4 ng/ml; n = 4.
The exposure of rats to saturated ether vapor for 2 min is widely used to provoke stress, and its effect on endogenous epinephrine and adrenocorticotropin secretion, two of the major mediators of stress response, is well known (25). The endogenous adrenergic secretory response to acute ether stress in conscious rats resulted in an immunoreactive pentapeptide circulating level of 7.0 ± 4.1 ng/ml;n = 4. Extraction and fractionation by cation-exchange HPLC of plasma samples obtained under pharmacological or acute stress-induced conditions showed that 56 ± 21%,n = 6, of the immunoreactive peptide fraction corresponds to free pentapeptide; the rest corresponds primarily to the SMR1-derived hexapeptide and undecapeptide. The immunoquantification of the pentapeptide plasma fraction could be assessed only if sampling and successive steps of extraction were carried out in the presence of EDTA or DTPA.
The physiological range of circulating pentapeptide in conscious adult male rats was, therefore, established to be 1–7 ng/ml.
Time Course of Plasma SMR1-Derived Pentapeptide Distribution and Elimination
The in vivo fate of infused pentapeptide in the circulation was investigated. Plasma pentapeptide and its metabolites were measured with time after a single injection of a physiological quantity of 110 ng tritiated pentapeptide into the circulation of two male rats. After reaching a maximal level within 2 min, the plasma pentapeptide concentration decreased rapidly, returning close to a basal level after 30 min (Fig. 3).
HPLC fractionations of plasma peptide extracts revealed that, in the bloodstream, 35% of the infused pentapeptide was metabolized within 4 min, and that proteolysis occurred from the amino-terminal part of the peptide. Plasma acidic pH treatment before Porapak Q extraction partially dissociated a pentapeptide-binding substance complex representing ∼45% of the circulating peptide fraction.
Elimination was investigated by measuring radioactivity excreted over the time course of the experiment and was estimated in anesthetized rats, at 6 pmol, eliminated through glomerular filtration, over a 60-min period. Approximately 80% of the urine radioactivity appeared to be pentapeptide metabolites.
These results suggest that distribution of circulating pentapeptide to the tissues was almost complete 30 min after injection. After this period, to allow for the total distribution of the peptide, we examined its cellular uptake at 60 min postinjection with physiological concentrations of [3H]peptide.
Cellular Localization of [3H]pentapeptide Binding Sites
The understanding of the function of SMR1-related peptide requires information about the identity of the cellular location of its binding sites within the target tissues identified above. Because the uptake of drugs or hormones can be influenced by dose, the significance of the selective cellular uptake of the pentapeptide was determined using physiological circulating concentrations of the tritiated molecule. The distribution volume for the pentapeptide in the male rat was calculated to be 35–40 ml/100 g body wt, which is similar to the extracellular fluid volume. Therefore, 5-wk-old rats received between 110 and 160 ng [3H]pentapeptide to reproduce physiological peptide plasma concentrations of 10-wk-old male rats for the following experiments:1) cellular localization of peptide binding sites and 2) regional and cellular saturation of peptide binding sites after coinjection of excess unlabeled peptide.
Light microscope autoradiographs of the kidney revealed the presence of silver grains confined preferentially within the deep inner cortex and the outer stripe of the outer medulla over the epithelial cells of the S3 segment of the straight portion of the proximal tubules. Dense silver grains were also seen within the S1 and S2 segments of the initial convoluted portion of these tubules (Fig.4 A). No specific cellular label was noticeable within the glomeruli or within the epithelia of distal and collecting tubules.
Within the glandular gastric mucosa, 60 min after [3H]pentapeptide injection, silver grains were exclusively distributed to the basal half of the gastric glands, over the chief or peptic cells (Fig.4 B). Within the pancreatic tissue, silver grains were selectively and homogeneously concentrated over the cells of acini (Fig. 4 C). No label was observed within the various ducts and islets of Langerhans. Contrary to expectation, within the submandibular target tissue, no selective cell association of label was identifiable.
Figure 4, E andF, illustrate a section of part of the thoracic vertebrae and the proximal end of tibia, respectively, both showing the trabecular bone. Within the bone tissue, the highest accumulation of silver grains occurred exclusively in the internal surface of the bone, in the spaces within marrow tissue, the trabeculae bone spaces. The intratrabecular spaces entrap bone cells, primarily the osteocytes, and their long cytoplasmic processes occupy the lacunae and canaliculi. Within these spicules of bone, the silver grains were denser over the canalicular layer than over the lacunar layer. Specific accumulation of silver grains was not noticeable either in the cartilagenous growth plate or in the hematopoietic marrow (Fig. 4,E andF). Figure4 D illustrates a section into the root of the rat upper incisor. In this dental tissue, silver grains were selectively concentrated over the entire dentin layer, along the length of the dentinal tubules of the canalicular system.
In displacement experiments, the cellular and total tissue distributions of [3H]pentapeptide were examined after 60 min after coinjection of 100-fold excess of unlabeled peptide. The large excess of cold-ligand concentration resulted in the almost complete displacement of [3H]pentapeptide uptake within the renal outer medulla and diffusion of label toward the inner medullary collecting ducts (Fig.5 A). Excess unlabeled peptide reduced binding to various extents within the glandular mucosa of the stomach, pancreatic and submandibular lobules, and long bone, with specific labeling percentages of 38–61, 37–55, 51–91, and 29–38%, respectively. The weak detectable reduction of radiolabeled peptide could be accounted for by the low abundance of receptor sites or the presence of significant radioactive degradation products distributed between the plasma and interstitial space, and may obscure saturable binding in vivo (31). In our experiment, it appears that the effectiveness in detection of saturable binding in vivo depends on the distribution of the peptide binding sites within the specific organs, either wide distribution (pancreatic and submandibular lobules) or narrow distribution (bones, stomach, kidney), and on the selectivity of the analysis methods used, i.e. total tissue count or regional difference analysis, respectively (Fig. 5 B, kidney, specific labeling percentage from 12 to 92%).
The basic concept of a functional hormone implies regulated synthesis and release into the circulation and selective, rapid, stable, and saturable uptake by target tissues due to the presence of specific hormone binding sites. We have previously established the physiological context (in the rat SMG) within which the androgen-regulated SMR1 precursor is processed to the final secretory products by a limited maturation pathway (24).
In the present study, using an in vivo labeling method coupled to quantitative β-radio imager analysis of whole rat body sections, we demonstrate that circulating SMR1-derived pentapeptide gains access to selective regions within the kidney, pancreas, SMG, bone, tooth, and stomach. The tissue uptake profiles have essential characteristics that allow specific binding sites to be identified in vivo: the binding is rapid (90 s after administration, at the latest), stable (240 min), selective, and saturable. We also demonstrate that this peptide hormone can bind to tissue receptor sites at adult male rat physiological circulating concentrations.
As supported by the analysis of quantitative β-radio imager and kinetics of the distribution and elimination of the pentapeptide in the male rat bloodstream, at 90 s and 3 min (circulating peptide distribution phase), the amount of pentapeptide in a tissue reflects the sum of the amounts in the plasma and interstitial space plus bound receptor sites. At 60 min (circulating peptide elimination phase) and later, the amount reflects the peptide binding sites-mediated sequestration. Therefore, the measure of selectivity and saturability of tissue and cellular uptake, realized 60 min after peptide administration, actually reflects specific binding sites for the pentapeptide. A rapid and stable distribution and accumulation of the peptide was demonstrated within the outer medulla of kidney, pancreatic lobules, glandular mucosa of the stomach, and periosteal and alveolar bone tissues as well as the incisor dentinal structure. The lack of early pentapeptide uptake by the inner bone matrix could be related to a substantially slower rate of exchange between the blood and the bone extracellular fluid compared with the exchange between the blood and the nonbone extracellular fluids (6).
In addition, the present study showed equivalent selectivity in the in vivo localization of binding sites-mediated sequestration for the pentapeptide between male and female rats, aged 5 wk. However, a reduced rate of peptide uptake appeared in the female. The decreased uptake capacity of most targets, except for the kidney and the SMG, could reflect a reduced number of binding sites for the pentapeptide in these tissues or an increased elimination rate of active peptide from the bloodstream in females. Metabolic and pharmacokinetic studies in the female rat bloodstream should clarify this point. At 5 wk (pubescence), the sexual dimorphism in rat SMG expression is appreciable, (i.e., the level of SMR1 mRNA in the male is about ten times more than in female SMG) (I. Rosinski-Chupin, C. Rougeot, and F. Rougeon, unpublished observations). It will be interesting to compare pentapeptide uptake efficiency between mature male and female rats, when synthesis and secretion of gonadal hormones and SMR1-related peptides is at a maximum (from 10-wk-old postpubertal rats). Such an approach requires the use of sialoadenectomized animals or an in vitro labeling method because it is essential to remove endogenous sources of circulating peptide (17).
We extended this study by identifying the cellular localization of the bound pentapeptide in each tissue in vivo. In this way the site of action can be deduced, an essential step in the determination of the role of the SMR1-derived peptide in male rats.
The present results provide direct evidence that pentapeptide binding sites are localized within specific portions of the male rat nephron, with a density of distribution in the area of the deep inner cortex and the outer stripe of the outer medulla and in particular over the epithelial cells of S3, S2, and S1 segments of the proximal tubules. Therefore, from a histological point of view, the findings give evidence for a role of the circulating pentapeptide in the regulation of renal function in adult male rats. The proximal convoluted tubule plays a major role in the reabsorption of Na+, , Cl−, Ca2+, , water, and organic solutes such as glucose and amino acids. The activity of most, if not all, renal epithelial transporting systems is hormonally regulated by steroids (gonadal and glucocorticoadrenal steroids) and peptide hormones (pancreatic, pituitary, and parathyroid hormones) (27). Most of the hormones that modulate tubule reabsorption and secretion processes act on membrane receptors and gain access to the cells from the blood (27). As a consequence, we are currently examining whether systemically administrated pentapeptide will promote modification, as an hormonal regulator, of hydromineral clearance in the rat in vivo.
The SMR1-derived pentapeptide may have direct effects on specific receptor sites or act indirectly by modulating the binding functions of hormones and other agonists that act via renal epithelial membrane receptors. In vitro molecular and pharmacological characterization of the receptor sites for SMR1-derived peptides needs to be investigated.
The visualization of pentapeptide binding sites within the internal area of bone, the trabeculae bone tissue, and within the periosteal bone surface, the periosteum, provides in situ evidence for a role for the pentapeptide in the regulation of bone remodeling activities. Although bone remodeling is a poorly understood process, numerous data indicate that this activity is ensured by various hormones, including steroids (androgens, estrogens, and glucocorticoids) and peptide hormones (parathyroid hormone, growth hormone, insulin growth factor-1, thyroxine, glucagon) derived from the blood supply to the bone (5). Furthermore, within the trabecular bone remodeling unit, which has the highest bone turnover rate and hormone responsiveness, the pentapeptide accumulates on the long cytoplasmic processes of osteocytes, the canaliculi. These tubular channel processes lie adjacent to osteocyte lacunae and open to extracellular fluid at the bone surface. They are involved in the deposition or resorption of calcium and phosphate ions that are present in the bone extracellular fluid and with the laying down of hydroxyapatite crystals. In addition, the periosteum is needed in bone regeneration during fracture repair (12). This finding suggests that SMR1-derived peptides may, as an hormonal modulator, contribute to the regulation of bone dynamics and turnover in the adult male rat.
In addition, SMR1-derived pentapeptide could also act as a mineral transport agent. However, its exclusive uptake by skeletal bone matrix (not by cartilagenous matrix) and renal proximal tubules (not by distal tubules) argues against such an hypothesis. It could also act as an activator or inhibitor of membrane or matrix enzyme activity important in skeletal and dental mineralization and mineral renal reabsorption. Such peptide-enzyme interactions would be expected to have high-affinity binding characteristics, giving the selective in vivo localization that we observed in the present study. Furthermore, the enzyme would have a restricted pattern of expression, which could coincide with the tissue distribution and the limited density of sequestration sites for the pentapeptide (∼0.1 fmol/mm2 tissue).
Evidence for the existence of pentapeptide binding sites localized within tubules of dentinal rat incisor was obtained. The layer of mature dentin is postulated to be involved in the initiation of the mineralization process of the tooth (3). Strikingly, it has been reported that the hormonal stimulation of fluid movement through these dentinal tubules may be dependent in part on parotid factors that are carried by the circulation to the teeth (15, 26).
Pentapeptide uptake by dentinal tissue and alveolar bone of the incisors, which are subject in the rat to continuous growth and rapid remodeling, respectively, provides additional evidence that the SMR1-derived peptide is involved in the regulation of mineral balance between skeletal, dental, and renal mineral transport and thus mineral homeostasis. Furthermore, in relation to male rat-specific behavioral characteristics, these data suggest that the androgen-regulated SMR1-derived pentapeptide may be a component, operating under stressful circumstances that lead to its systemic secretion, of a feedback loop to regulate the cascade side effects on mineral balance, thereby satisfying mineral homeostatic requirements. In relation to female rat-specific physiological characteristics, this tight regulatory mechanism may manage mineral homeostasis during late pregnancy and/or lactation. Indeed, lactation places extraordinary demands on mineral homeostasis (5) and coincides with a marked increase of mature peptide content in the female SMG (unpublished data).
The gastric chief cells and pancreatic acinar cells of males are also targets for the pentapeptide, suggesting that the SMR1-derived peptide may have a functional role in modulating the synthesis and/or secretion in both of the zymogen-secreting cells and/or secretion of the fluid and electrolytes in the acinar cells. The secretion of gastric and pancreatic digestive enzymes is highly regulated, and significant amounts of the enzymes are released only on stimulation of the zymogenic cells, as occurs during feeding (reviewed in Refs. 11 and 13). In addition, the major mechanism for regulation of enzyme secretion is generally held to be stimulation by secretagogues. Secretagogue receptors for a number of peptide hormones have been described on these cells, including the secretin/vasoactive intestinal peptide and the cholecystokin families and specifically gastrin-releasing peptides, tachykinins for acinar cells (reviewed in Refs. 11 and 13). The demonstration of pentapeptide binding sites within these exocrine cells supports the hypothesis of a regulatory role of SMR1-derived peptide in the systemic control of early digestive processes. To reinforce this hypothesis, we are presently examining whether feeding or stressful starving behavior will promote the systemic secretion of the pentapeptide.
Chromatography characteristics of circulating and tissue-bound pentapeptide revealed that the transport and the uptake of the peptide involves a complex molecular species, including a cation mineral element. The characterization of both the acid- and organic solvent-proof pentapeptide complex is currently underway. As is generally observed for all major hormones, SMR1-derived pentapeptide is anchored to binding molecules in the plasma to prevent degradation and/or to facilitate transport.
In conclusion, our data lead us to postulate that the circulating SMR1-derived pentapeptide is primarily involved in the hormonal control of mineral balance between at least four systems: the bone, the kidney, the tooth, and the bloodstream. Mineral imbalance can occur in response to acute or chronic stress circumstances, including intraspecies fighting and feelings of pain, thirst, starvation, and harmful temperatures (10). The circulating pentapeptide may be a component of a feedback loop that regulates, in adult male rats, the cascade response to environmental stress within which mineral intake or excretion is modified, thereby controlling mineral homeostasis.
Pentapeptide is specifically taken up by the mineralized dentin, trabecular and alveolar bone, the remodeling units that have the highest bone turnover rate and hormone responsiveness, as well as by the renal mineral transporting epithelia of male rats.
Therefore, attention will be focused on elucidating the physiological roles of the peptide originating from the SMG and prostate in the biology of the targets. In particular, we will investigate biological properties mediated by SMR1 maturation peptides on mineral metabolism and transport, concentrating on bone and dentin matrix formation and mineral deposition.
Of importance will be efforts to explore the direct actions of the SMR1-derived pentapeptide, in particular its ability to potentiate the effect of hormones and growth factors that are known to be highly involved in bone metabolism and mineral homeostasis. The molecular and pharmacological characterization of pentapeptide receptor sites will increase our understanding of its purpose.
From a evolutionary point of view, it is difficult to imagine that the male rat has developed its own tight regulatory system for controlling mineral homeostasis under behavioral stress situations. An implicit assumption of this research is that the endocrine loop between the sites of production of SMR1-related pentapeptide and sites of action is a “security system” adapted to the environmental, behavioral, and physiological characteristics of mammals. The knowledge of the physiological and eventual pathological states that control or affect SMR1 maturation peptide synthesis and release in relation to mineral transport and deposition rate is needed to confirm this hypothesis.
The authors are pleased to acknowledge Drs. Brecheriou (anatomo-pathologie, Hopital St. Louis, Paris), Verbavatz (Departement Biologie Cellulaire et Moléculaire, Commisariat à l’ Energie Atomique, Gif-sur-Yvette), and Huerre (histo-pathologie, Institut Pasteur) for helpful histological identification. The assistance of Dr. G. Charpak (Ecole Supérieure de Physique et Chimie Industrielles, Paris) in β-radio imager quantification of whole rat body sections and J. C. Benichou (biologie moleculaire, Institut Pasteur) in resin sectioning are gratefully acknowledged. We especially thank Drs. G. Langley, B. Laoide, and I. Rosinski-Chupin for critical reading of this manuscript.
Address for reprint requests: C. Rougeot, Unité de Génétique et Biochimie du Développement, Département d’Immunologie, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris Cedex 15, France.
This study was supported by grants from the Institut Pasteur and Direction des Recherches, Etudes, et Techniques (contract 93/113 DRET).
- Copyright © 1997 the American Physiological Society