Basal Cytosolic Calcium Research Articles

Platelet basal cytosolic calcium: The influence of plasma factors in cirrhosis

Background: Platelet cytosolic calcium is necessary for normal platelet function, and may reflect intracellular signalling in vascular smooth muscle cells. Methods: The cytosolic calcium of platelets from patients with cirrhosis and control subjects was measured in contact with plasma from either source, using FURA 2AM. Results: The basal cytosolic calcium of patients with cirrhosis was found to be significantly lower than that of control subjects (95.7±10.0 cf. 128.0±7.8 nmol/l; p=0.02). When platelets from control subjects were incubated for 45 min with freshly obtained plasma from patients with cirrhosis, the control platelet cytosolic calcium fell to concentrations similar to those of patient platelets (93.0±7.8 nmol/l; p<0.005). Such an effect was not observed if patient serum or plasma that had previously been frozen was used. When patient platelets were incubated with fresh control subject plasma, the platelet cytosolic calcium increased (165.4±19.9 nmol/l; p=0.01). Conclusion: These results indicate that a plasma-borne factor, sensitive to freezing, is responsible for the abnormalities of platelet calcium signalling noted in cirrhosis.

Extracellular chloride regulates mesangial cell calcium response to vasopressor peptides.

The role of extracellular chloride in the regulation of mesangial cell calcium responsiveness to vasopressor peptides was explored. First, the components of vasopressor-stimulated calcium signaling were defined in rat mesangial cells cultured on coverslips and preloaded with fura 2. By spectrofluorometry, manganese uptake (reflecting divalent cation channel activation) was observed by quenching of fura 2, or intracellular cytosolic calcium concentration was calculated by dual-excitation ratiometric measurement. In cells depolarized with KCl (45 mM), enhanced manganese uptake or increased cytosolic calcium were inhibited with verapamil (10 microM). Pretreatment of mesangial cells with verapamil reduced the sustained calcium level in response to endothelin-1 (0.1 microM) by 65 +/- 6% (means +/- SE, n = 12) and to vasopressin (1 microM) by 62 +/- 12% (n = 8). Perforated cell patch-clamp measurement confirmed that endothelin-1 stimulated a sustained increase in cytosolic calcium or divalent cation entry only in the presence of simultaneous depolarization. In chloride-free buffer (chloride replaced with impermeant anions), sustained calcium response to endothelin-1 was reduced by 72 +/- 8 (n = 8) and by 65 +/- 4% (n = 8) in the presence of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (55 microM). In chloride-free buffer, cytosolic calcium (unstimulated) increased to > 200 nM by 30 min. These data indicate that reduced extracellular chloride increases mesangial cell basal cytosolic calcium and decreases the transient and sustained cytosolic calcium response to vasopressor peptides.

Cyclic adenosine 3',5'-monophosphate-independent regulation of cytosolic calcium in Sertoli cells.

We have examined the cAMP-independent regulation of cytosolic calcium concentration in rat Sertoli cells using the effect of vasoactive hormones, known as testicular paracrine regulators operating via the non-cAMP pathway, on cytosolic calcium. Calcium concentrations were estimated with dual excitation fluorimetry, using freshly isolated, fura-2/AM-loaded cells. No increase in the cellular cAMP concentration was detected after stimulation with angiotensin II (AII), vasopressin, PGF2 alpha, or atrial natriuretic peptide. Whereas both AII and vasopressin evoked a rise in cytosolic calcium from a basal level of 81.4 +/- 4 to 142.5 +/- 18 and 154.4 +/- 11 nM, respectively, PGF2 alpha had only a minimal effect (98 +/- 5 nM), and atrial natriuretic peptide no effect (86.6 +/- 9 nM). The effect of AII on calcium was blocked by the the selective AT2, but not by the AT1, receptor antagonist, indicating the selective presence on Sertoli cells of AT2 AII receptor. Similarly, the vasopressin-induced calcium response was blocked by vasopressin V1, but not by V2 receptor antagonist, consistent with the presence of V1 receptor subtype in these cells. Removal of extracellular calcium or blockade of calcium channels did not inhibit the calcium increase due to AII and vasopressin, suggesting the involvement of intracellular calcium. Thapsigargin increased the basal cytosolic calcium concentration to 137 +/- 10 nM. Depletion of intracellular calcium stores with thapsigargin before stimulation with AII or vasopressin abolished both the AII-mediated and the vasopressin-mediated calcium rise in the presence as well as the absence of extracellular calcium, indicating that the increase in calcium is predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. This study indicates that calcium homeostasis of Sertoli cells might also be regulated by cAMP-independent metabolism apart from the well known cAMP-dependent pathway. Furthermore, our findings support the idea that angiotensin and vasopressin might be important paracrine regulators of Sertoli cells functions.

Intracellular calcium concentration impairment in hepatocytes from thioacetamide-treated rats. Implications for the activity of Ca 2+-dependent enzymes

Methods/Results: Thioacetamide induced a severe perivenous necrosis followed by a hepatocellular regenerative response, when administered in a single dose of 6.6 mmol/kg to rats. As (Ca 2+) i plays an important role in both toxic cell killing and cell proliferation, the disturbances in the basal cytosolic calcium as well as the levels of Ca 2+ sequestered in the endoplasmic reticulum were determined in hepatocytes isolated at 0, 12, 24, 48 and 72 h after thioacetamide administration. The basal Ca 2+ increased progressively, reaching a maximum at 24 h of the intoxication (205%, p<0.001), while the microsomal sequestered Ca 2+ decreased at 24 h to 16% ( p<0.001) when compared with untreated controls. Changes in the activity of glycogen phosphorylase a paralleled those of basal free calcium and showed the maximum value also at 24 h (291%; p<0.001). Moreover; there was a close association in time between the basal concentration of Ca 2+ and the inhibition of microsomal Ca 2+-dependent ATPase activity. Conclusions: The significant decrease in the levels of GSH and protein thiols indicates that oxidative stress is involved in thioacetamide-induced cell injury, but these decreases did not precede changes in cytosolic Ca 2+ level. In the sequence of events leading to hepatic cell injury and regeneration, thioacetamide mobilized hepatic (Ca 2+) i via inhibition of microsomal Ca 2+-ATPase which may have activated Ca 2+-dependent mechanisms involved both in cell death and in acute mitogen response.

Serotonin-induced increases in platelet cytosolic calcium concentration in depressed, schizophrenic, and substance abuse patients

Exaggerated intracellular calcium responses to challenges with serotonin (5-HT) have been reported in depression. In our studies, consistent with previous reports, patients with depression exhibited an exaggerated increase in 5-HT-stimulated intracellular calcium concentration ([Ca++]i). Basal cytosolic calcium was elevated in both calcium-free and 1 mM calcium media in depressed patients. the increased responsiveness to 5-HT was seen in both conditions. Patients with schizophrenia and substance abuse did not differ from normal controls. The 5-HT response was correlated with diastolic blood pressure (r = 0.33, p = 0.02): however, this association did not fully account for the exaggerated [Ca++]i responses in the depressed group. These findings suggest that exaggerated increases in [Ca++]i in response to serotonin are a characteristic of depressed patients not shared with schizophrenic and substance abuse patients. The relationship of depression to hypertension, two conditions that share abnormalities of calcium homeostasis, warrants further study.

Simvastatin releases Ca2+ from a thapsigargin-sensitive pool and inhibits InsP3-dependent Ca2+ mobilization in vascular smooth muscle cells.

Simvastatin (SV), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity inhibits migration and proliferation of vascular smooth muscle cells (SMC). To investigate whether these effects of SV are related to inhibition of cell calcium mobilization, cultured SMC obtained from rat aorta were loaded with Fura-2 to determine the basal cytosolic free calcium levels ([Ca2+]i) and the agonist-stimulated Ca2+ mobilization. SV (20 mu M) transiently increased cytosolic free calcium, an effect that depends mainly on intracellular calcium release (68%). This effect of SV was markedly reduced (75%) by thapsigargin, an inhibitor of the Ca2+ ATPase of inositol 1,4,5-triphosphate (InsP3)-sensitive calcium pools. Incubation of cells with SV (15 min) inhibited the mobilization of Ca2+ by angiotensin II, platelet-derived growth factor, and vasopressin (IC50 = 5 mu M). SV did not affect inositol trisphosphate (InsP3) levels or modify its generation by angiotensin II (Ang II) and vasopressin. Furthermore, in saponin-permeabilized cells, SV abolished the release of calcium by 2,3-dideoxy-InsP3. SV reduced the effect of thapsigargin on InsP3-sensitive stores by 67%, suggesting that SV depletes these calcium pools. The inhibitory effect of SV on calcium mobilization was prevented by coincubation of cultured cells (24 h) with 1 mM mevalonic acid, the product of HMG-CoA reductase activity. These results support the notion that SV inhibits [corrected] the migration and proliferation of SMC by directly affecting cell Ca2+.

The role of plasma in the disturbance of basal cytosolic calcium in platelets from patients with cirrhosis: [formula omitted]. Liver Research Laboratories, Department of Medicine, Royal Infirmary, Lauriston Place, Edinburgh. Scotland

The role of plasma in the disturbance of basal cytosolic calcium in platelets from patients with cirrhosis: [formula omitted]. Liver Research Laboratories, Department of Medicine, Royal Infirmary, Lauriston Place, Edinburgh. Scotland

Effects of thapsigahgin, an intracellular CA 2+ pump inhibitor, on insulin release by rat pancreatic B-cell

This is the first report as to the effects of thapsigargin (Tg), an inhibitor of intracellular Ca 2+ pumps, on insulin release by pancreatic B-cells. Tg does not alter basal insulin release by the isolated islets, with 3 mM glucose. However, it potentiates high glucose-induced insulin release: potentiation of the first phase response is dose-related in a concentration range of 1.3–40 μM. In isolated B-cells, Tg causes a minimal rise in basal cytosolic free calcium concentration ([Ca 2+] i) and eliminates high glucose-induced initial lowering of [Ca 2+] i. Tg does not alter glucose oxidation by the islets and the islet insulin content. An elimination of glucose-induced sequestration of Ca 2+ into Tg-sensitive intracellular pool(s) is considered to be the cause of Tg potentiation of glucose effect on insulin release.

Bombesin does not stimulate pepsinogen release in isolated gastric chief cells.

Bombesin (BN)-related peptides, such as gastrin-releasing peptide (GRP), have been shown in vivo to stimulate release of pepsinogen. However, whether this is due to a direct interaction with chief cells is not clear. To clarify this we prepared isolated chief cells (> 90% pure) from guinea pig stomach. BN, GRP, or neuromedin B (NMB), at concentrations up to 1 microM, did not stimulate pepsinogen release or affect the stimulation caused by vasoactive intestinal peptide (VIP) (100 nM) or CCK-8 (10 nM), respectively. In addition, BN, GRP, or NMB at a concentration of 1 microM did not increase cAMP nor did they alter the increase in cAMP caused by VIP or secretin. BN (1 microM) did not alter basal cytosolic calcium [Ca2+]i or affect the increase in [Ca2+]i caused by CCK-8 (1 microM). Furthermore, BN, GRP, or NMB at a concentration of 1 microM did not increase the generation of inositol phosphates (IP) or alter the increase in [3H]IP1, [3H]IP2, or [3H]IP3, caused by CCK-8 (1 microM) or carbachol (1 mM). Binding studies demonstrated no saturable binding of either [125I][Tyr4]BN or [125I][D-Tyr0]NMB using experimental conditions where binding with other peptide ligands to other receptors on chief cells is seen. We conclude that BN-related peptides do not interact directly with specific receptors on chief cells to stimulate or alter stimulated pepsinogen secretion, increase the breakdown of inositol phosphates, or alter [Ca2+]i or cAMP.

Alterations in basal and serotonin-stimulated calcium permeability and vasoconstriction in atherosclerotic aorta.

Hypersensitivity to vasoactive stimuli, a common finding in atherosclerotic arteries, is thought to play an important role in the pathology of arterial and coronary vasospasm and may be a factor in myocardial ischemia and infarction. While this phenomenon is well documented, the underlying mechanism is unknown. The present study used isometric force measurements coupled with 45Ca2+ and Fura 2-AM techniques in aortic smooth muscle to probe transmembrane calcium movements and cytosolic calcium levels in an attempt to determine their relation to altered vasomotion in a rabbit model of dietary atherosclerosis. Following 10 weeks of cholesterol feeding (2%), basal (unstimulated) calcium influx was augmented 1.5-fold in atherosclerotic segments with no change in basal calcium efflux. Serotonin-stimulated calcium uptake was increased 1.9-fold in atherosclerotic segments and was accompanied by a fivefold increase in serotonin vasoconstrictor sensitivity and a 1.4-fold increase in serotonin-stimulated calcium efflux. Endothelial denudation did not alter either force generation or 45Ca2+ movements in serotonin-stimulated segments. In arterial smooth muscle cells dispersed from atherosclerotic vessels, basal and serotonin-stimulated cytosolic calcium levels were augmented approximately 2.3-fold and twofold, respectively. These findings contribute to our understanding of the cellular defects in calcium metabolism, which may ultimately explain the cellular basis of serotonin hypersensitivity in atherosclerotic arteries and certain arterial vasospastic syndromes in this disease state.

Hepatic Ito cells contain calcium channels: increases with transforming growth factor-beta 1.

Ito cells (fat-storing cells) have been implicated in mechanisms of liver fibrosis, and transforming growth factor-beta 1 is a key factor that stimulates collagen production by Ito cells. Moreover, Ito cells are reported to possess contractile proteins and to contract with ligands. We recently reported the presence of L-type voltage-operated Ca2+ channels in Kupffer cells. In this study, we examined whether Ito cells contain Ca2+ channels and also evaluated the effect of transforming growth factor-beta 1 on Ca2+ channels. Cytosolic free calcium concentration was measured in individual cultured Ito cells with the fluorescent Ca2+ indicator dye fura-2. Partial replacement of extracellular Na+ with K+ caused an increase in cytosolic free calcium, presumably as a result of transmembrane Ca2+ influx. Basal cytosolic free calcium levels were around 40 to 50 nmol/L in both control and transforming growth factor-beta 1-treated cells. In transforming growth factor-beta 1-treated cells, cytosolic free calcium increased in response to K+ at values as low as 10 mmol/L, whereas untreated cells did not respond. Half-maximal increases in cytosolic free calcium in transforming growth factor-beta 1-treated cells were observed with 63 +/- 6 mmol/L K+. With 100 mmol/L K+, intracellular free calcium increased around fourfold above basal values in transforming growth factor-beta 1-treated cells but was only increased about twofold in untreated controls. We conclude that this increase in cytosolic free calcium occurs by way of voltage-operated calcium channels; it did not occur in the absence of extracellular calcium and cannot be explained by Na+/Ca2+ exchange mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

High Glucose Attenuates Peptide Agonist-Evoked Increases in Cytosolic Free [Ca2+] in Rat Aortic Smooth Muscle Cells

Incubation of cultured rat aortic smooth muscle cells (ASMCs) in a medium containing high glucose concentrations (25 mM) did not affect the basal cytosolic free calcium ([Ca2+]i) but led to significant reductions in peak [Ca2+]i response evoked by arginine vasopressin, angiotensin II, and endothelin-1 (ET-1). This was observed in both the presence and absence of extracellular Ca2+. Maintenance of rat ASMCs in a medium containing mannose (an osmotic control for high glucose) did not affect either the basal or peptide agonist-evoked increase in [Ca2+]i. However, pretreatment with either the nonselective protein kinase C (PKC) inhibitor staurosporine or the selective PKC inhibitor 2,6-diamino-N-([1-(1-oxotridecyl)-2 piperidinyl] methyl) hexanamide reversed the attenuating effect of high glucose on peak [Ca2+]i response evoked by ET-1. Also, short-term incubation of ASMCs with the active phorbol ester, phorbol 12-myristate 13-acetate, led to a reduction in peak [Ca2+]i response to all three agonists, whereas the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, had no such effect. Although high-glucose treatment of rat ASMCs led to significant reductions in the maximal number of binding sites to the extent of 39% of [125I]ET-1 specific binding, no significant differences in the affinity (Kd approximately 110 pM) characteristics were evident between control and high-glucose treatment groups. It is proposed that incubation of rat ASMCs with high glucose enhances the de novo synthesis of diacylglycerol and activates membrane-bound PKC and that this, in turn, impairs agonist-mediated intracellular Ca2+ mobilization.

Intracellular signal transduction for vasoactive peptides in hypertension.

Increased peripheral resistance is the hallmark of hypertension. It may result in part from exaggerated vascular reactivity of resistance arteries. Some changes in density of surface receptors for different vasoconstrictors and vasorelaxants have been described that could play a role in physiological findings in hypertension. Smooth muscle cells of resistance arteries have increased cytosolic free calcium concentration in some models of experimental hypertension, which may contribute to enhance vascular responses. Exaggerated response of the inositol phosphate-calcium pathway has been demonstrated after stimulation with some vasoconstrictor agents such as norepinephrine, angiotension II, and vasopressin. In contrast, responses to the potent vasoconstrictor peptide endothelin-1 are either normal (in spontaneously hypertensive rats) or blunted (in deoxycorticosterone-salt hypertension). In the latter case, endothelin receptor density, inositol phosphate and diacylglycerol generation, and cytosolic calcium responses agree with blunted response of blood vessels. Increased basal cytosolic calcium and exaggerated sensitivity of myosin light chain to calcium may be mechanisms underlying increases in sensitivity of signal transduction in smooth muscle in some models of hypertension. However, in general, signal transduction of receptors for vasoconstrictors appears to be blunted rather than exaggerated, except for responses to angiotensin II. Altered structure of resistance arteries (remodeling) may be a mechanism that, even in presence of blunted intracellular signal transduction, may result in enhanced pressor responsiveness of blood vessels in hypertension.

Blood pressure inversely correlates with thrombin-evoked calcium rise in platelets.

Earlier investigations showed a positive correlation between basal cytosolic free calcium in human platelets and blood pressure; however, recent studies have failed to show this relation. We undertook the present work to examine which platelet cytosolic calcium parameters (namely, cytosolic calcium in resting or stimulated states in calcium-containing and calcium-free media) present the least variability and best correlation with blood pressure. We studied 17 healthy white men on three different occasions separated by 1- and 4-week intervals. Their manual and ambulatory automated 24-hour blood pressure measurements were correlated with cytosolic calcium in resting and stimulated (thrombin-treated) fura 2-loaded platelets. The following cytosolic calcium parameters were measured in 1 mmol/L calcium and calcium-free media: basal cytosolic calcium, peak thrombin-evoked cytosolic calcium, and post-transient cytosolic calcium 5 minutes after thrombin treatment. The highest and lowest coefficients of variation were respectively shown by the basal cytosolic calcium (22.8%) and peak thrombin-evoked cytosolic calcium (10.1%) in calcium medium. Basal cytosolic calcium did not correlate with any of the blood pressure parameters. Of the cytosolic calcium parameters, peak thrombin-evoked cytosolic calcium in calcium medium demonstrated consistent (negative) correlations with blood pressure, with better correlations shown with diastolic than systolic blood pressure of both automated and manual blood pressure readings. Peak thrombin-evoked cytosolic calcium in calcium medium showed similar correlations with nighttime and daytime automated blood pressure measurements. There were no correlations between peak thrombin-evoked cytosolic calcium in calcium-free medium and blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II but not to endothelin-1

To determine the differential calcium responses to two vasoconstrictor peptides, angiotensin II (Ang II) and endothelin-1, in vascular smooth muscle cells derived from mesenteric arteries from young and adult normotensive and hypertensive rats. Effects of Ang II and endothelin-1 on cytosolic free calcium concentration in primary cultured unpassaged single vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto (WKY), Wistar and spontaneously hypertensive rats (SHR) aged 3, 9 and 17 weeks were examined microphotometrically using fura-2 methodology. Basal cytosolic free calcium concentration was significantly increased in cells from SHR aged 9 and 17 weeks compared with cells from age-matched WKY and Wistar rats. Ang II and endothelin-1 significantly increased cell cytosolic free calcium in all rat groups at all ages. Responses to low concentrations of Ang II (1 nmol/l) were significantly higher in cells from SHR aged 9 and 17 weeks than in age-matched controls. This was confirmed in cells from rats aged 17 weeks with full concentration-response curves, which also showed that the pD2 for Ang II for WKY rats was significantly different from that of SHR. In cells from SHR at all ages Ang II-stimulated cytosolic free calcium remained persistently high, whereas in cells from WKY and Wistar rats basal levels were reached within 100 s after the maximal response. Low concentrations of endothelin-1 elicited significantly lower cytosolic free calcium responses in cells from SHR aged 17 weeks compared with age-matched controls. The time course of cytosolic free calcium responses to endothelin-1 were similar in the groups. In primary cultured unpassaged mesenteric vascular smooth muscle cells from adult SHR, cytosolic free calcium concentration responses to Ang II are enhanced, whereas responses to low concentrations of endothelin-1 are slightly reduced. The differential effects of these two vasoconstrictor peptides may contribute to their relative roles in modulating vascular smooth muscle cell cytosolic free calcium in SHR.

Activated complement directly modifies the performance of isolated heart muscle cells from guinea pig and rat

The complement system has been implicated in the pathogenesis of cardiovascular disorders including ischemia and atherosclerosis. Selective deposition of C5b-9, the membrane attack complex of complement, has been histochemically documented in human myocardium early after reperfusion of ischemic areas and in infarcted zones. However, functional sequelae of the C5b-9 complex binding to myocardial cells have not been identified. Insertion of C5b-9 complexes into the membrane of other cell types can generate transient changes in membrane permeability in the absence of cell lysis. We demonstrate in beating isolated adult guinea pig and rat cardiac myocytes that human derived C5b-9 can transiently augment in a dose-dependent manner both basal cytosolic calcium concentration and calcium transients, resulting in a temporary increase in contractility. If similar changes occur in human heart cells in vivo, they could significantly affect myocardial performance and contribute to functional abnormalities seen in ischemia and other pathological conditions associated with complement activation.

Protein kinase C inhibitors enhance endothelin-1 and attenuate vasopressin and angiotensin II evoked [Ca2+]i elevation in the rat cardiomyocyte.

Primary cultures of neonatal rat cardiomyocytes were pretreated for 16 h with either nonselective (staurosporine, 100 nM) or selective (NPC15437, 20 microM) protein kinase C (PKC) inhibitors. These inhibitors did not affect the basal cytosolic free calcium, [Ca2+]i, level (106 +/- 12 nM) as determined by fura-2 fluorescence methodology. Both agents significantly enhanced the maximal [Ca2+]i responses to endothelin-1 (ET-1) and attenuated the peak [Ca2+]i responses to arginine vasopressin and angiotensin II. They did not alter the EC50 values of any of these agonists. Since depletion of [Ca2+]o led to only partial attenuation of the enhanced response to ET-1 in the treatment groups, it is likely that PKC inhibition results in an exaggerated intracellular mobilization of Ca2+ to ET-1. It is concluded that PKC modulates agonist(s)-evoked intracellular Ca2+ mobilization and that the nature of regulation is governed by the agonist.

Calcium, phosphoinositide, and 1,2-diacylglycerol responses of blood vessels of deoxycorticosterone acetate-salt hypertensive rats to endothelin-1.

In previous studies a decreased responsiveness to endothelin-1 (ET-1) of conduit arteries and resistance vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats was found in comparison with uninephrectomized controls. Decreased isometric force, number of receptors, and inositol phosphate accumulation were reported in the DOCA-salt animals. In the present study effects of ET-1 on cytosolic free calcium, inositol phosphates, and 1,2-diacylglycerol were investigated in blood vessels of DOCA-salt hypertensive rats. Basal cytosolic free calcium, measured with the fluorescent dye fura-2, was 201 +/- 41 nmol/l in mesenteric arteries of DOCA-salt rats and 45 +/- 9 nmol/l in uninephrectomized controls (p less than 0.01). The maximal response of cytosolic free calcium (to 30 nmol/l ET-1) was 176 +/- 22% of the basal value for DOCA-salt and 242 +/- 6% for uninephrectomized rats (p less than 0.05). The concentration giving 50% of the maximum response was 9.0 and 6.5 nmol/l for DOCA-salt rats and controls, respectively. Inositol phosphate production after stimulation with 100 nmol/l ET-1 in the presence of LiCl was lower by at least 30% (p less than 0.01) in both aorta and mesenteric arteries of DOCA-salt hypertensive versus control rats. Basal levels of diacylglycerol in aorta were similar in DOCA-salt rats and in controls and did not respond to a 100 nmol/l ET-1 stimulation in the DOCA-salt rats, in contrast to the increase found in the control uninephrectomized rats (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase C responses in cells from spontaneously hypertensive rats.

We tested the hypothesis that increased systemic vascular resistance in spontaneously hypertensive rats may be secondary to enhanced phospholipase C activity in response to vasoconstrictor stimuli. Activation of phospholipase C by angiotensin II (Ang II), thromboxane A2, arginine vasopressin, and endothelin-1 was compared in cultured glomerular mesangial cells and mesenteric vascular smooth muscle cells taken from 13- to 14-week-old hypertensive and normotensive Wistar-Kyoto rats (blood pressure, 185 +/- 1 versus 135 +/- 2 mm Hg). Phospholipase C was assessed by measuring cytosolic free calcium and by the accumulation of radiolabeled inositol phosphates. Basal cytosolic calcium did not differ between mesangial cells taken from both strains but was greater in smooth muscle cells from hypertensive rats (210.1 +/- 8.2 versus 149.2 +/- 4.7 nM). The responsiveness of cytosolic calcium and inositol phosphate accumulation to Ang II was significantly enhanced in mesangial cells from hypertensive rats (10(-7) M Ang II: peak increase of calcium, 1,266 +/- 181 versus 603 +/- 93 nM; percent increment of inositol phosphates at 1 minute, 266 +/- 26 versus 98 +/- 10%). Vascular smooth muscle cells from hypertensive rats, when compared with normotensive rats, showed a similar augmentation of Ang II-stimulated intracellular calcium and inositol phosphates. Thromboxane A2-induced enhancement of intracellular calcium and inositol phosphate accumulation in vascular smooth muscle cells was also greater in hypertensive animals. However, the responses to vasopressin and endothelin in mesangial or vascular smooth muscle cells did not differ between the normotensive and hypertensive animals. There was no significant difference in Ang II receptor number and affinity between hypertensive- and normotensive-derived mesangial cells. We conclude that genetically increased blood pressure in rats may be secondary to enhanced post-receptor signaling in glomerular mesangial cells activated by Ang II and to enhanced signaling in vascular smooth muscle cells stimulated by either Ang II or thromboxane A2.

Oxytocin at physiological concentrations evokes adrenocorticotropin (ACTH) release from corticotrophs by increasing intracellular free calcium mobilized mainly from intracellular stores. Oxytocin displays synergistic or additive effects on ACTH-releasing factor or arginine vasopressin-induced ACTH secretion, respectively.

The potency of oxytocin (OT) in evoking ACTH secretion by isolated, superfused rat adenohypophyseal corticotrophs and its enhancement by CRF and arginine vasopressin (AVP) were analyzed. Each secretagogue effectively released ACTH from adenohypophyseal cells when added separately in pulsatile fashion in physiological concentrations based on hypophyseal portal blood (OT, 10 nM; AVP, 0.5 nM; CRF, 0.1 nM). OT released ACTH at concentrations as low as 1 nM. Moreover, a dose-response relationship up to 10 microM was revealed. Combinations of a constant amount of CRF (0.1 nM) with increasing concentrations of OT exerted a synergistic effect on ACTH release. In contrast, OT given in various concentrations in combination with AVP (0.5 nM) produced an additive effect on ACTH release. To study the mechanism of action of OT on ACTH secretion, cytosolic free calcium levels in single pituitary cells exposed to OT or AVP were measured using the calcium-sensitive fluorescent indicator Fura-2. Corticotrophs among mixed adenohypophyseal cell types in the primary cultures were identified by immunocytochemistry. More than 500 cells were individually stimulated with OT or AVP. Basal cytosolic free calcium levels ranged between 80-130 nM free calcium. The addition of 100 nM OT or 1 microM AVP increased the cytosolic free calcium concentration within 3 sec to values ranging from 500-800 nM. An increase in intracellular calcium ranging from 200-500 nM due to OT could still be observed after extracellular calcium depletion. Taken together, our data demonstrate that physiological concentrations of OT stimulate ACTH secretion, independent of the other ACTH secretagogues, by mobilizing calcium mainly from intracellular stores.