Changes of renin isoelectric heterogeneity after acute and chronic stimulation of renin secretion

Changes in the multiple forms of renin secreted or stored in vitro by renal cortical slices were studied in rats made hypertensive with deoxycorticosterone, adrenalectomized rats, and rats fed a high or low salt diet. Renal slices from normal rats were also incubated with angiotensin II, vasopressin, and verapamil. Aliquots of incubation media were subjected to isoelectric focusing, and the six forms of renin were quantified and expressed as a percentage of the total renin activity recovered from the gel. The results showed that chronic and acute stimulation of renin secretion produced a similar modification of the isoelectric focusing profile, consisting of an increased proportion of renin forms with the more acidic isoelectric points. The change in the proportions of the more acidic renin forms was greater with chronic stimulation than that after stimulation with verapamil. However, chronic and acute inhibition or reductions of the rate of renin secretion did not modify the renin profile. We suggest that the progression in the shift of secreted renin forms to those with the more acidic isoelectric points correlates with the intensity or duration of stimulation of renin secretion. These data support the hypothesis that different pools of renin exist and are altered differently by chronic and acute stimulation of renin secretion.     

Coexistence of renin and angiotensin II in epitheloid cell secretory granules of rat kidney

Summary The distribution of renin and angiotensin II (ANG II) in juxtaglomerular epitheloid cells of control and adrenalectomized rats was studied, using specific antisera and the protein A-gold technique in Lowicryl- and glycol methacrylate-embedded tissue.The matrix of virtually all mature secretory granules of epitheloid cells contains not only renin, but also ANG II. On adrenalectomy, the concentration of both renin and ANG II in the granule internum increases markedly, as indicated by the density of the immunolabel.Given the coexistence of renin and ANG II in the granule matrix, it is quite probable that, with each secretory event, a certain amount of ANG II is released together with renin. Further experiments will have to show if this amount of ANG II cosecreted with renin is sufficient to elicit immediate local intrarenal actions.ANG I, as well as angiotensinogen and converting enzyme, were not found in epitheloid cells. It is therefore inferred that ANG II is not generated intracellularly, but within the extracellular space and subsequently taken up by pinocytosis and incorporated into the secretory granules of epitheloid cells.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Coexistence of renin and cathepsin B in epithelioid cell secretory granules

Summary Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin B-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin B is suggested to be involved in the activation of renin prior to secretion.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Coexistence of renin and cathepsin B in epithelioid cell secretory granules

Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin B-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin B is suggested to be involved in the activation of renin prior to secretion.     

Cathepsin D coexists with renin in the secretory granules of juxtaglomerular epithelioid cells

Summary Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin-and cathepsin D-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin D is suggested to be involved in the regulation of the granular renin stores available for secretion.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Cathepsin D coexists with renin in the secretory granules of juxtaglomerular epithelioid cells

Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin D-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin D is suggested to be involved in the regulation of the granular renin stores available for secretion.     

Rapid refilling of neurosecretory terminals with secretory granules after an acute stimulus to the isolated neural lobe of the rat

Summary Quantitative ultrastructural techniques were used to study changes in the distribution of intracellular organelles in neurosecretory terminals of the rat neurohypophysis during recovery from a depolarising stimulus in vitro. The volumetric density of neurosecretory granules which, in profiles of nerve terminals sectioned through the area of contact with the basal lamina, was decreased as a result of stimulation, returned to control values within 2 h after cessation of stimulation. We conclude that, even in the absence of the cell body and action potentials propagated from it, granules can migrate quickly within the terminal region of the neurosecretory axons to refill a depleted compartment.     

Quantitative electron microscopic studies on the kinetics of secretory granules in G-cells

Summary Ultrastructural studies of secretory granules of rat antral G-cells and measurement of serum gastrin level were performed under the condition of fasting and administration of alkaline solution into the stomach. On electron micrographs, no qualitative difference was observed among those experimental groups. However, morphometrical analysis revealed significant quantitative differences. The population density of secretory granules of the rats treated once with alkali first increased and then decreased reaching that of the fasted group, while that of the repeatedly treated group remained nearly equal to the maximum value. The average sectioned surface area of secretory granules tended to decrease for 1.5h after the stimulation but the difference was not significant among those groups.From the results obtained at present, responding to chemical stimulation such as pH changes in the antrum, it seems probable that not only exocytosis but also migration of secretory granules from supra- and/or para-nuclear portion to the basal portion of the cell occurs rapidly in G-cells and that both these processes are inhibited immediately by antral acidification. Moreover, the present results apparently indicate that under the condition of no antral acidification G-cells have a capacity of secreting gastrin for a fairly long time, such as 4–8 h, responding to adequate stimulus. These findings are strongly suggestive of the existence of a capacious pool of granules in the supra- and/or para-nuclear cytoplasm or of fairly speedy production of secretory granules in the Golgi area.The author wishes to express thanks to Prof. R. Furihata, Department of Surgery, and Prof. T. Nagata, Department of Anatomy, Shinshu University School of Medicine, for their constant interest and guidance, and to Dr. F. Iida, Department of Surgery, who has followed the course of this work throughout     

Morphological and functional changes in cell junctions during secretory stimulation in the perfused rat submandibular gland

To examine the influence of cholinergic and beta-adrenergic agents on paracellular transport, we applied confocal microscopy and freeze-fracture to the isolated, perfused submandibular gland of the rat. By confocal microscopy, perfusion of lucifer yellow through an arterial catheter, revealed a bright fluorescence in the basolateral spaces of acini, but not in the intercellular canaliculi. However, addition of isoproterenol on carbachol stimulation, induced lucifer yellow fluorescence in intercellular canaliculi. This finding indicates that isoproterenol is capable of opening the paracellular route. The tight junction strands surrounding intercellular canaliculi were visualized using freeze replicas. Fixation was carried out both by vascular perfusion with Karnovsky's solution and by metal contact rapid freezing with liquid helium. In the chemically-fixed specimens, the strand particles of tight junctions formed 2-5 lines at the P-face along most of the apical portion at rest. With carbachol/isoproterenol stimulation, the strand particles rearranged with free ends and terminal loops. In the rapidly frozen specimens, the strand particles were arranged more irregularly even in the resting state. The meshwork of strands became more disheveled and interrupted during carbachol/ isoproterenol stimulation. The present findings led us to conclude that: 1) the beta-adrenergic agent, isoproterenol, can open the paracellular transport. 2) in the rapidly frozen specimen, the tight junction strand particles are arranged roughly and become disheveled and interrupted during stimulation by carbachol/isoproterenol. These findings may be related to rearrangement of subcellular structures, especially of the actin filament network.     

Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery

We utilized quantitative electron microscopic immunogold labeling procedures to follow changes in the intragranular content of five secretory proteins of the rat submandibular gland (SMG) during and after chronic treatment with the beta-adrenergic agonist isoproterenol (IPR). Labeling intensities (gold particles/microns2) of acinar cell secretory granules for mucin and glutamine/glutamic acid-rich proteins, major secretory proteins of the normal SMG, showed opposite responses to IPR. Labeling intensities increased for mucin and decreased for glutamine/glutamic acid-rich proteins immediately after IPR injections began, then rapidly returned to control levels after cessation of IPR treatment. SMG Protein C immunoreactivity, found in both acinar and intercalated duct granules, was less affected by IPR. However, opposite changes in labeling intensity were observed between acinar and intercalated duct granules. Labeling intensities for proline-rich proteins, IPR-inducible secretory proteins, increased only after 10 days of stimulation and maintained a high level even after cessation of drug treatment. Type 2 cystatin, another IPR-inducible protein, increased gradually with chronic IPR treatment and decreased slowly during the recovery phase. These results suggest that chronic beta-adrenergic stimulation affects the expression of genes for several rat SMG secretory proteins in a different manner.     

Studies on the molecular organization of rat insulin secretory granules

Secretory granule-enriched fractions prepared from isolated rat islets of Langerhans, previously labeled in culture for 18 h with [3H]leucine, have been lysed and separated into pH 5.4 soluble and insoluble fractions by zonal sucrose gradient centrifugation. A high proportion of both labeled and immunoreactive rat insulins I and II were recovered in the insoluble granule core fraction in the expected ratio of approximately 60/40, respectively. Essentially equivalent amounts of the rat C-peptides on a molar basis were recovered in the granule supernatant fractions. The proportion of labeled proinsulin in the granule core fraction was less than 2% relative to insulin, while the soluble fraction contained about 8%, which probably arose mainly from disrupted proinsulin-rich noncrystalline prosecretory vesicles. Electron microscopic examination of the granule core fraction revealed large numbers of well preserved crystalline cores exhibiting typical dimensions and regular internal spacings of normal mature rat beta-granule inclusions. These results provide direct biochemical evidence that the beta-granules are nonuniform in composition with the insulin contained mainly in a crystalline state in the electron-dense central inclusions while the C-peptide is dissolved in the fluid bathing the crystalline hormone. The significance of this structural organization of the beta-granule is discussed.     

Elementary granules,small vesicles and exocytosis in the rat neurohypophysis after acute haemorrhage

Summary Neural lobes of rats subjected to severe acute haemorrhage under sodium pentobarbitone anaesthesia were examined electron microscopically and the ultrastructure compared with that in anaesthetised and unanaesthetised controls. Changes in the localisation and numerical distribution of elementary granules and small vesicles in the neurohypophysial nerve endings of bled rats were consistent with the occurrence of exocytosis. The occurrence of exocytotic profiles was observed more frequently in freeze-etched tissue samples as compared with the material fixed for conventional electron microscopy. The ratio of small vesicles: elementary granules was shown to be significantly increased (P<0.005) in the nerve endings of neural lobes from bled rats. Equally, the numbers of exocytotic profiles related to 1000 m² of neurohypophysial tissue area were significantly greater (P<0.005) in bled rats.The study was supported by Medical Research Council of Canada. The authors are grateful to Dr. W. Costerton, Biology Department, The University of Calgary, for use of facilities for freeze-etching, and to Miss Y. Carter for technical assistance.Research Associate, Consejo Nacional de Investigationes Cientificas y Tecnicas, Argentina.Associate, Medical Research Council of Canada.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.     

Semi-automatic morphometric analysis of secretory granules in the cells of the rat adenohypophysis

The authors carried out a morphometric analysis--using a semi-automatic system for image analysis--of the secretory granules in the cells of the Wistar rat adenohypophysis. They evaluated the mean diameter (CIRCLE D), maximum diameter (MAX D), the factor of circular form (PE FORM) and the harmonic factor of form (HAR FORM). They also computed the coefficient of correlation between CIRCLE D and PE FORM and the percentual distribution of the values according to mean diameter.