Osmotic and nonosmotic control of vasopressin release.

While the existence of an osmotic control for vasopressin (AVP) release has been long recognized, development of a sensitive immunoassay has allowed for better understanding of factors affecting the threshold and sensitivity of AVP release. Individual variation, genetic, environmental, and species differences, and the nature of the solute providing the osmotic stimuli can significantly affect the release of the hormone by altering the threshold and/or the sensitivity of the osmoreceptor. In addition to the hypothalamic osmoreceptor, AVP secretion is also controlled by an anatomically separate pathway which is responsive to nonosmotic stimuli. It appears that both low-pressure (left atrial) and high-pressure (carotid and aortic) receptors via the parasympathetic pathways provide the major nonosmotic pathway for vasopressin release. Such pathways are activated in response to acute systemic hemodynamic changes, stress, and hypoxia. The precise interaction between osmotic and nonosmotic AVP release remains to be clarified. A model of osmotic and nonosmotic interactions, based on available electrophysiologic studies, is presented and its clinical implications are discussed.     

Role of brain acetylcholine in vasopressin release during osmotic stimulation and hemorrhage

Summary There is considerable evidence to suggest that there is a cholinergic link in the neural control of vasopressin release, but the precise role for this link has not been adequately demonstrated in the intact animal. We have, therefore, examined in conscious unrestrained rats the effects of central cholinergic blockade on the stimulation of vasopressin release by increased plasma osmotality (iv infusion of 2.5 M NaCl at 0.1 mg/kg body weight · min for 30 min) and by decreased blood volume (2 successive hemorrhages of 10% of blood volume each). The vasopressin responses to these stimuli were unaffected by either intracerebroventricular (icv) atropine (10 g; muscarinic blockade) or icv hexamethonium (10 g; nicotinic blockade) in doses which block the vasopressin responses to icv cholinergic agonists. The implications of these findings are discussed.On leave from the Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, JapanOn leave from the Third Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan     

Pulsatile arginine vasopressin release from the rat hypothalamo neurohypophyseal system during osmotic stimulation

Arginine vasopressin (AVP) was released in vitro in a pulsatile pattern from the hypothalamo-neurohypophyseal system (HNS) and from the hypothalamus during continuous hyperosmotic stimuli with NaCl or fructose. No significant difference was found in the AVP pulse frequency between the two kinds of hyperosmotic agents. AVP was released from the HNS in a dose-related manner under NaCl stimulation. When the neural lobe was stimulated with NaCl or fructose, a clear AVP pulse pattern was not apparent. Urea failed to evoke a significant AVP release from the neural lobe or HNS. A stepwise increase in NaCl stimulation from 5 to 25 mEq induced a AVP response from the HNS and hypothalamus similar to that under constant stimulation at 25 mEq NaCl. This phenomenon was also found with fructose or sucrose. These results suggest that AVP release from the HNS during continuous osmotic stimulation has a pulsatile pattern regardless of the hyperosmotic substance or osmotic pressure. This AVP release accurately reflects the physiological function of the hypothalamus without modulation in the neural lobe. These results also suggest that the total amount of AVP was related to the osmotic pressure or the osmotic substance but that the frequency of the pulse release was not, moreover, that the AVP release depends not only on the absolute osmotic pressure, but also on the changing rate of osmotic pressure.     

Role of intracellular calcium in histamine release from rat mast cells

An aqueous extract of Panax Ginseng C.A. Meyer (G.S.) was prepared by boiling crushed G.S. roots in water. The extract obtained was adjusted to 125 mg G.S. per ml and was administered orally to mice for 5 to 6 days at the daily dose of 10, 50 and 250 mg G.S. per kg or was added to cultures of mouse spleen cells at concentrations varying between 0.25 and 8 mg G.S. per ml. The average total ginsenoside content of the G.S. roots used was determined by HPLC analysis and found to be 0.58%(w/w).Treated mice responded with enhanced antibody formation to either a primary or a secondary challenge with sheep red cells. The effects were dose-dependent. At the highest dose regimen, the primary IgM response was increased by 50% and the secondary IgG and IgM responses were increased by 50 and 100%, respectively. An even more pronounced effect was obtained with natural killer cell activity which was enhanced between 44 and 150% depending on the effector-to-target cell ratios used in the assay. In vitro, G.S. showed two main effects, an inhibition of stimulated and spontaneous lymphocyte proliferation at high, but not cytotoxic concentrations and an enhancement of interferon production particularly in non-stimulated spleen cells.The immunostimulating effects obtainedin vivo are in agreement with the stimulation of interferon production observedin vitro. The inhibition of lymphocyte proliferation, however, cannot be reconciled with the immunostimulatory action of G.S. observedin vivo.     

Role of intra- and extracellular calcium in histamine release from rat peritoneal mast cells

The present study provides evidence for a number of calcium pools important in histamine secretion from the mast cell. Firstly, calcium loosely bound to the cell membrane, and in rapid equilibrium with the extracellular environment, may be utilized for histamine release induced by most secretagogues. Secondly, all inducers are able to mobilize deeply buried or internal stores of calcium to initiate exocytosis. Finally, calcium bound to regulatory sites in the membrane may modulate the secretory process. Removal of calcium from the latter sites by brief treatment with chelating agents markedly enhances the secretory response in the absence of extracellular calcium, probably by facilitating the mobilization of bound stores of the ion. Saturation of these sites in the presence of excess calcium inhibits the release process and may restrict influx of the cation.     

Role of intra- and extracellular calcium in histamine release from rat peritoneal mast cells

The present study provides evidence for a number of calcium pools important in histamine secretion from the mast cell. Firstly, calcium loosely bound to the cell membrane, and in rapid equilibrium with the extracellular environment, may be utilized for histamine release induced by most secretagogues. Secondly, all inducers are able to mobilize deeply buried or internal stores of calcium to initiate exocytosis. Finally, calcium bound to regulatory sites in the membrane may modulate the secretory process. Removal of calcium from the latter sites by brief treatment with chelating agents markedly enhances the secretory response in the absence of extracellular calcium, probably by facilitating the mobilization of bound stores of the ion. Saturation of these sites in the presence of excess calcium inhibits the release process and may restrict influx of the cation.     

Calcium and osmotic stimulation in renin release from isolated rat glomeruli

The effects of changes in osmolality and calcium concentration on renin release (RR) from isolated superfused rat glomeruli were studied.The undisturbed RR followed a first order fall with a half-time of about 100 min (n=45). Changes in the osmolality between 270 and 350 mOsm/kg resulted in dosedependent changes in the RR rates. Hypoosmotic treatment stimulated the RR transiently, whereas hyperosmotic treatment produced a sustained inhibition. the dose-response relationship was log-linear between 270 and 320 mOsm/kg. A decrease in osmolality of 20 mOsm/kg gave proportional increases in RR irrespectively of the RR rate preceding the stimulus.Removal of calcium stimulated the RR by 10 times (n=5,p<0.001) and a subsequent decrease in osmolality of 20 mOsm/kg stimulated the RR proportionally to that observed in the series containing 2 mM calcium. A decrease in osmolality was able to stimulate RR (n=5.5,p<0.05) even when the calcium concentration in the medium was simultaneously raised from 0 to 2 mM.A hyperosmotic Ringer (+300 mOsm/kg), inhibited RR to very low levels. A subsequent removal of external calcium was now unable to stimulate the release (n=5.5). In a less hyperosmotic Ringer (+50 mOsm), the RR was inhibited, but a removal of external calcium now stimulated RR.It is suggested that the osmosensitivity of the RR process reflects a waterflux-driven fusion of secretory granules with the cell membrane, and that calcium affects an intragranular equilibrium between aggregated, osmotically inert granule content and dissolved, osmotically active granule content.Some of the results were presented in preliminary form at Vth European Colloquium on Renal Physiology, Frankfurt, June 1985, abstract no. 43     

Phasic firing enhances vasopressin release from the rat neurohypophysis

1. Isolated rat neural lobes were incubated in vitro and electrically stimulated to release vasopressin. The released vasopressin was assayed using a radioimmunoassay and there was a reasonably good correlation (r = 0.81) between results obtained with this assay and those obtained by bioassay with the rat blood pressure method.2. Regular stimulation at frequencies of 5, 10 and 20 Hz released progressively more vasopressin and the release could be blocked by addition of tetrodotoxin to the incubation medium.3. Stimulation with pulse patterns derived from tape recordings of phasically firing units in the supraoptic nucleus of dehydrated rats released more vasopressin than the same number of pulses regularly spaced in time. In the range 2-8 pulses/sec vasopressin release was related to the pulse frequency within the bursts (r = 0.90) and the number of short (< 100 msec) interpulse intervals (r = 0.92). Vasopressin released per pulse increased over the frequency range 3-6 pulses/sec, but above 6 pulses/sec vasopressin release per pulse tended to diminish.4. We conclude that phasic firing of vasopressin neurosecretory cells may enhance vasopressin release in vivo and that an important factor in determining release is the number of short interspike intervals.     

Regulation of activity-dependent dendritic vasopressin release from rat supraoptic neurones

Magnocellular neurones of the hypothalamus release vasopressin and oxytocin from their dendrites and soma. Using a combination of electrophysiology, microdialysis, in vitro explants, and radioimmunoassay we assessed the involvement of intracellular Ca²⁺ stores in the regulation of dendritic vasopressin release. Thapsigargin and cyclopiazonic acid, which mobilize Ca²⁺ from intracellular stores of the endoplasmic reticulum, evoked vasopressin release from dendrites and somata of magnocellular neurones in the supraoptic nucleus. Thapsigargin also produced a dramatic potentiation of dendritic vasopressin release evoked by osmotic or high potassium stimulation. This effect is long lasting, time dependent, and specific to thapsigargin as caffeine and ryanodine had no effect. Furthermore, antidromic activation of electrical activity in the cell bodies released vasopressin from dendrites only after thapsigargin pretreatment. Thus, exposure to Ca²⁺ mobilizers such as thapsigargin or cyclopiazonic acid primes the releasable pool of vasopressin in the dendrites, so that release can subsequently be evoked by electrical and depolarization-dependent activation. Vasopressin itself is effective in inducing dendritic vasopressin release, but it is ineffective in producing priming.     

Role of vasopressin in rat distal colon function

The specific role of vasopressin in colonic crypt function and its possible synergistic action with aldosterone were studied. Sprague-Dawley rats fed a high-Na⁺ (HS; 150 m m NaCl) or a low-Na⁺ (LS; 150 μ m NaCl) diet were deprived of water or infused with vasopressin, and some animals were treated with specific vasopressin receptor subtype V₁ and V₂ antagonists. The expression of the epithelial Na⁺ channel (ENaC), α-smooth muscle actin (α-SMA) and aquaporin-2 (AQP-2) were determined by immunolocalization in distal colonic mucosa. The pericryptal Na⁺ concentration was determined by confocal microscopy, using a low-affinity Na⁺-sensitive fluorescent dye (sodium red) and crypt permeability was measured by the rate of escape of fluorescein isothiocyanate-labelled dextran (10 kDa) from the crypt lumen into the pericryptal space in isolated rat distal colonic mucosa. A high plasma concentration of vasopressin raised α-SMA expression in the pericryptal sheath ( P < 0.05), increased the pericryptal Na⁺ accumulation in this space ( P < 0.01) and caused a reduction of crypt wall permeability ( P < 0.01). All these effects were reversed by selective blockade of V₁ and V₂ receptors. No synergistic effects with aldosterone were observed. Dehydration and vasopressin infusion increased AQP-2 expression in distal colonic mucosa ( P < 0.05). This action of vasopressin was prevented by tolvaptan, a specific V₂ receptor antagonist ( P < 0.05). It is concluded that vasopressin has trophic effects in the rat distal colon, increasing pericryptal myofibroblast growth which affects crypt absorption, and these effects are independent of the presence of aldosterone.     

Chloride and magnesium dependence of vasopressin release from rat permeabilized neurohypophysial nerve endings

The role of Cl- and Mg+ ions has been studied on the secretory mechanism leading to the release of vasopressin from digitonin permeabilized nerve endings isolated from the rat neurohypophysis. Secretion was triggered by challenging the permeabilized nerve endings with 1.1 microM free Ca2+. Magnesium enhances secretion and its maximal effect occurred at a concentration of about 2 mM. Further increase of this divalent cation concentration however led to an inhibition of secretion. Chloride ions are necessary for the final steps in exocytosis and this effect of Cl- was inhibited by the chloride channel antagonist N144. It is concluded that in neurosecretory nerve endings magnesium and chloride ions are crucial components for exocytosis to occur.     

Chronic osmotic stimulation reduces vasopressin but not synaptophysin content in rat neurohypophysis

The content of synaptophysin, a vesicular integral membrane protein of neurons and endocrine cells, and that of vasopressin was measured in neurohypophyses of rats during chronic osmotic stimulation. The animals received 2% NaCl in their drinking water for up to 4 days. Synaptophysin content of neurohypophyses was determined using quantitative immunoblotting, vasopressin content was measured by radioimmunoassay. Salt loading caused a decrease in the content of vasopressin to about 15% of that of control animals, whether expressed per neurohypophysis or relative to the total tissue protein. In contrast, no change was found in the synaptophysin content. Taken together with published evidence of changes in the relative numbers of the hormone-containing neurosecretory granules (NSGs) and the microvesicles (MVs) under the conditions of chronic osmotic stimulation, these results strongly indicate the surface density of synaptophysin on NSGs to be significantly lower than its surface density on MVs.     

Effect of age on calcium release from mouse calvaria in tissue culture

The effect of age on calcium release in organ culture was studied using calvaria from 5-, 14-, 30-, and 75-day-old mice. The results demonstrated a different and characteristic calcium release pattern over the 14-day culture period for untreated calvaria of different ages. Since calcium release in all cultures was abolished by procedures such as boiling, multiple freezing and thawing, maintaining cultures in an oxygen-free gas phase, or maintaining cultures in a non-nutritive medium, it was concluded that the calcium release from older calvaria was due to a cell-mediated process. Histological observations demonstrating the presence of Howship's lacunae and active osteoclastic resorption confirmed that calcium release from older calvaria was due to an active bone resorption process. Parathyroid hormone did not substantially alter the general pattern of calcium release exhibited by different aged calvaria. However, it tended to exaggerate the magnitude of the response. Indomethacin and dexamethasone inhibited calcium release from untreated 5- and 75-day-old calvaria suggesting that prostaglandin biosynthesis was involved in the calcium release process. Direct measurements of PGE₂ and PGI₂ released into the culture medium gave results consistent with this hypothesis, although it is conceivable that indomethacin and dexamethasone might have influenced calcium release by other mechanisms.     

Interaction of calcium and somatostatin in the control of prolactin and somatotropin release from a primary culture of rat adenohypophyseal cells

Neuroscience and Behavioral Physiology -     

Role of calcium in stretch-induced release and mRNA synthesis of natriuretic peptides in isolated rat atrium

 To investigate the role of Ca²⁺ in stretch-induced synthesis and release of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) isolated superfused rat atria were stretched by raising intra-atrial pressure. The immunoreactive (ir-) ANP and BNP concentrations were analysed by radioimmunoassay and the corresponding mRNA levels were quantified by Northern blot and dot blot analyses. Stretch-induced ir-ANP release and a rise in BNP mRNA levels increased at high (3.0 mM) compared to low (0.5 mM) extracellular Ca²⁺ concentration ([Ca²⁺]_o). Moreover, the adaptation of stretch-induced ir-ANP release was dependent on [Ca²⁺]_o. Atrial BNP mRNA levels were increased by stretch also in non-paced, electrically silent atria, where voltage-activated Ca²⁺ channels are not activated. The stretch-induced rise in BNP mRNA was blocked by gadolinium (80 μM), but not by the L-type channel blocker diltiazem (3.0 μM). This study indicates that both the stretch-secretion coupling of ir-ANP release and the pressure-stimulated synthesis of BNP mRNA are Ca²⁺-dependent processes. Gadolinium inhibits the stretch-stimulated rise in BNP mRNA levels in contracting and non-contracting atria, which is similar to its ability to block stretch-activated ir-ANP release, suggesting the involvement of Ca²⁺-permeable stretch-activated channels. Received: 29 April 1996 / Received after revision and accepted: 17 June 1996     

Histamine release from saponin-permeabilized rat mast cells by calcium

The first effect of receptor activation on the mast cell surface, initiating histamine secretion, is an increase in the cytosol Ca²⁺ concentration. It should then be possible to induce histamine secretion by calcium alone, if the calcium permeability of the cell membrane could be increased without any significant interference with the physiological cell functions. This was achieved in the present study by adding low concentrations of saponin (0.005% and 0.001% w/v) to the medium. When calcium was added to the saponinpermeabilized cells, around 40% histamine release occurred with 0.25 mM extracellular calcium (free Ca²⁺ 0.15 mM). The release was inhibited by antimycin A (1 M). Transmission electron microscopy showed formation of vacuoles containing granules stripped of their membranes, which characterize a secretory response. The observations are consistent with a limited increase in the calcium permeability of the cell membrane for a brief period. There was apparently an increase in the cytoplasmic calcium concentration, which acted through calmodulin, since the histamine release induced by calcium from the permeabilized mast cells could be inhibited by a calmodulin-antagonist, mepacrine (10–30 M).