Platelet Cytosolic free Calcium Before and After Antihypertensive Treatment in Perinephritis Hypertension of the Rabbit

Cytosolic free calcium concentration ([Ca²⁺]_i) in platelets has been reported to be elevated in human essential hypertension, to be positively correlated with blood pressure and to decrease with blood pressure reduction. However, some groups have been unable to confirm these findings in either humans or hypertensive rats. We have examined the relationship between platelet [Ca²⁺]_i and blood pressure in the perinephritis model of hypertension in the rabbit. In addition, the effects of both acute and chronic treatment with verapamil or prazosin were studied. Mean arterial pressure, heart rate and platelet [Ca²⁺]_i were measured before and after treatment. Platelet [Ca²⁺]_i was measured by the Quin 2 fluorescence technique. Platelet [Ca²⁺]_i was similar for the normotensive and hypertensive rabbits, and no correlation between platelet [Ca²⁺]_i and blood pressure was observed. None of the antihypertensive treatments produced a lowering of platelet [Ca²⁺]_i. Therefore we conclude that platelet [Ca²⁺]_i is unlikely to be a universally useful index of [Ca²⁺]_i in vascular smooth muscle of resistance vessels.     

Platelet calcium handling is different in rats with salt-dependent and spontaneous forms of genetic hypertension

Increased platelet cytosylic free calcium concentration ([Ca²⁺]₁) has been demonstrated in both human essential hypertension and spontaneous hypertension of the rat. The present study was designed to extend the investigation on platelet Ca²⁺ handling to two models of salt-dependent genetic hypertension (Sabra and Dahl rat strains). No major [Ca²⁺]₁ elevation was seen in salt hypertensive SBH Sabra or SS/Jr Dahl rats. This contrasts with the data obtained in Lyon hypertensive rats (a spontaneous form of genetic hypertension) in which basal platelet [Ca²⁺]₁ was clearly increased and correlated positively with diastolic blood pressure. In these two strains, basal platelet [Ca²⁺]₁ correlated with pulse pressure but not with diastolic pressure. The absence of a significant relationship between platelet [Ca²⁺]₁ and diastolic pressure in both Sabra and Dahl rats indicates that, at least in young rats with developing salt hypertension, platelet cytosolic calcium need not reflect calcium changes occuring in the vascular smooth muscle of resistance arterioles. In contrast to the high values seen in Lyon hypertensive rats, the [Ca²⁺]₁ rise induce by thrombin was unchanged in salt-sensitive SS/Jr Dahl rats and substantially reduced in hypertension-prone SBH rats (irrespective of salt intake). The initial rate of thrombin-induced Mn²⁺ entry through receptor-operated Ca²⁺ channels was similar in SBN and SBH as well as in SR/Jr and SS/Jr rats kept on a low-salt diet but was reduced by high salt intake in platelets of salt-resistant (SBN and SR/Jr) animals only. Since platelets of Lyon hypertensive rats are also characterized by greater initial rate of thrombin-induced Mn²⁺ entry, this parameter was always higher in rats with established, hypertension compared to their respective normotensive controls. Our study demonstrated that alterations of platelet Ca²⁺ handling are different in salt-dependent than in spontaneous forms of gentic hypertension.     

Nisoldipine blocks the increase of intracellular free calcium-ion concentration associated with elevated sodium–lithium countertransport activity in erythrocytes in patients with NIDDM

To understand the mechanism by which elevated sodium–lithium countertransport activity (SLC) associates with increased intracellular free calcium-ion concentration ([Ca²⁺]_i), we investigated the relationship between SLC and the effects of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) and a Ca²⁺-channel blocker, nisoldipine, on [Ca²⁺]_i in erythrocytes from 48 patients with non-insulin-dependent (Type 2) diabetes mellitus (NIDDM). There was a significant correlation between SLC and [Ca²⁺]_i. Nisoldipine in the incubation medium significantly decreased [Ca²⁺]_i, and there was a significant positive correlation between SLC and the degree of [Ca²⁺]_i decrease. When the [Ca²⁺]_o was elevated, [Ca²⁺]_i was significantly increased, but nisoldipine almost completely suppressed this increase of [Ca²⁺]_i. There was a significant positive correlation between SLC and the degree of the suppression. These data suggest that elevated SLC correlates with increased [Ca²⁺]_i, and that the increased [Ca²⁺]_i might be due to the increased Ca²⁺ influx through a dihydropyridine-sensitive Ca²⁺ pathway. © 1997 by John Wiley & Sons, Ltd.     

Fluorescent Indicators Give Biased Estimates of Intracellular Free Calcium Change in Aggregating Platelets: Implication for Studies with Human von Willebrand Factor

ABSTRACT: The ratiometric fluorescent indicators Fura-2 and Indo-1 are considered optimal probes for monitoring intracellular free calcium concentration ([Ca²⁺]_i). Unique problems arise, however, in studying [Ca²⁺]_ichanges induced in platelets by von Willebrand factor (vWF). Binding of native multimeric vWF causes extensive platelet aggregation, and is reported to evoke a gradual [Ca²⁺]_iincrease. The present investigation examined the reliability of platelet [Ca²⁺]_imeasurements in these circumstances. Ristocetin-mediated binding of vWF to human platelets promoted a slow rise in Fura-2 fluorescence ratio. Fura-2 extrusion contributed substantially to this rise, unless blocked by probenecid. Despite this precaution, the platelets were invariably contaminated slightly with extracellular indicator. As aggregation progressively reduced the number of platelets in the spectrofluorometer beam, through settling of the larger aggregates, such extracellular Fura-2 contributed proportionately more to the observed fluorescence. This extraneous signal accounted completely for the fluorescence ratio increase, and apparent [Ca²⁺]_irise, in response to native multimeric vWF. The same problem arose with Indo-1, whereas the single wavelength indicator Fluo-3 showed the opposite pattern of apparent [Ca²⁺]_ichanges. Thus, none of these indicators provides reliable data on [Ca²⁺]_isignals in aggregating platelets. Use of a dimeric form of vWF eliminated the problem of platelet aggregates settling out of suspension, but also virtually abolished the [Ca²⁺]_iincrease. These observations may explain some of the inconsistencies among previous investigations of vWF-induced calcium signaling. Moreover, similar problems may arise in studies with other adhesive proteins.     

Increased Intracellular Ca2+ is Not Coinherited With an Inferred Major Gene Locus for Hypertension (ht) in the Spontaneously Hypertensive Rat

Hypertension is characterized by a complex mode of inheritance, consisting of the accumulation and interaction of major and minor genes. The existence of a single major gene locus (ht) has been demonstrated in the backcross analysis of spontaneously hypertensive rats (SHR) and normotensive Donryu rats. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) determines the tonus of vascular smooth muscle. It has been hypothesized that abnormal Ca²⁺ transport is an inheritable trait with profound influence on the development of hypertension.Backcross analysis between SHR and Donryu rats was performed to demonstrate ht and to dissect polygenic hypertensive traits through ht and abnormal intracellular Ca²⁺ metabolism. Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca²⁺]_i in platelets were significantly greater in SHR than in Donryu and F₁ rats. The backcrossed rats were distributed into two clusters on a scattergram of blood pressure versus [Ca²⁺]_i, demonstrating the existence of ht. The blood pressure level was correlated with thrombin-stimulated [Ca²⁺]_i in each cluster.Increased [Ca²⁺]_i in platelets was not coinherited with ht and was considered to be a minor inheritable hypertensive trait discriminated from ht. Therefore, [Ca²⁺]_i in platelets is an inadequate marker for searching ht.     

Endothelin-Induced Renal Vasoconstriction and Increase in Cytosolic Calcium in Renal Vascular Smooth Muscle Cells

This study was designed to investigate the effects of the potent vasoconstrictor, endothelin, on renal hemodynamics in rats in vivo, and in addition, to measure intracellular calcium ion ([Ca²⁺].) in monolayers of renal vascular smooth muscle cells in culture using the fura-2 method. Endothelin (1 nmol) dramatically decreased renal blood flow from 7.0±0.5 ml/min to 2±6±1±0 ml/min, whereas it increased mean arterial pressure from 100±2 mmHg to 113±7 mmHg. These alterations persisted over 20 minutes in conscious and almost unrestrained rats. Endothelin (10^?8 -10^?7 mol/l) immediately increased [Ca²⁺]_i, although the increase by endothelin (10^?9 mol/l) was relatively slow. The increase persisted in the presence of 1 mmol/l extracellular calcium. In the absence of extracellular calcium, only a small, transient increase of [Ca²⁺]_i was observed. These results indicate that endothelin produces renal vasoconstriction and increases the [Ca²⁺]_i in cultured renal vascular smooth muscle cells. The latter effect is dependent mainly on extracellular calcium.     

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: Effects of extracellular calcium and atrial natriuretic factor

Summary The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca²⁺]_i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca²⁺]_i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca²⁺]_i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca²⁺]_i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca²⁺]_i in calcium-replete buffer.     

Different features of Ca2+ oscillations in differentiated and undifferentiated hepatocyte doublets

Cytosolic free Ca²⁺ ([Ca²⁺]_i) oscillations are postulated to play a critical role in cellular proliferation. By using doublets of normal rats (NR) and those 18 hours after two-thirds hepatectomy (PHR), we investigated cytosolic free Ca²⁺ ([Ca²⁺]_i) responses in liver regeneration. Normal rat hepatocyte doublets that retain their bile canaliculi are polarized and well differentiated. PHR doublets, which also retain their bile canaliculi, were characterized as undifferentiated by (1) decreased canalicular secretion of fluorescein-isothiocyanate-labeled glycocholate; (2) increased labeling index of hepatocytes in BrdU staining (∼30%); and (3) impaired transfer of fluorescent dye injected into one cell of the pair to the other. Addition of phenylephrine to NR and PHR doublets in the presence of extracellular Ca²⁺ resulted in [Ca²⁺]_i oscillations or a nonoscillatory-sustained increase in [Ca²⁺]_i, followed by a gradual return to the baseline. Extracellular Ca²⁺ was not required for [Ca²⁺]_i, oscillations but was necessary for a sustained increase in [Ca²⁺]_i. Simultaneous addition of prazosin, al-receptor blocker, to doublets immediately abolished these [Ca²⁺]_i, responses. The [Ca²⁺]_i level in each of the adjacent cells was synchronous in sustained increase in [Ca²⁺]_i, but asynchronous in [Ca²⁺]_i oscillations. As the phenylephrine concentration was increased (1 to 100 μmol/L), oscillations were replaced by a sustained increase in [Ca²⁺]_i in NR doublets. In contrast, in PHR doublets, oscillations remained, whereas the frequency of oscillations increased in a dose-dependent manner. These results indicate that the mechanisms of phenylephrineevoked [Ca²⁺]_i responses are different in differentiated and undifferentiated doublets and that the frequency modulation of [Ca²⁺]_i oscillations may be involved in the intracellular signal transduction in the cellular proliferation process during liver regeneration.     

A link between polymorphonuclear leukocyte intracellular calcium,plasma insulin,and essential hypertension

BackgroundIntracellular ionized calcium ([Ca²⁺]_i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca²⁺]_i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca²⁺]_i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP).MethodsThe PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca²⁺]_i was measured by flow cytometry in isolated cells stained with Fluo-3.ResultsThe EH PMNs showed significantly increased [Ca²⁺]_i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca²⁺]_i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca²⁺]_i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04).ConclusionsThis study adds PMN to previously described cells exhibiting elevated [Ca²⁺]_i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca²⁺]_i and plasma insulin levels, together with the fact that elevated [Ca²⁺]_i mediates PMN priming, suggest that elevated [Ca²⁺]_i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.     

Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Effects of Erythropoietin on Endothelin-1 Synthesis and the Cellular Calcium Messenger System in Vascular Endothelial Cells

A rise in blood pressure is the main side effect of erythropoietin (EPO) treatment in patients with renal anemia. The mechanisms, however, by which EPO may cause hypertension are still unclear. We therefore investigated the effects of EPO on endothelin (ET) synthesis and cytosolic free calcium concentration ([Ca²⁺]_i) in vascular endothelial cells. Porcine endothelial cells were isolated from thoracic aorta, pulmonary artery, and vena cava. Studies were performed with cells of the first subculture. ET concentrations were measured radioimmunologically. Changes in [Ca²⁺]_i were determined with the fluorescent probe fura-2. Cytotoxicity was assessed by sodium 3′-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assay. ET synthesis was similar in cells of different vascular origins and was time-dependent, reaching approximately 2 pmol ET/mg protein within 12 h of incubation. EPO (12 to 200 U/mL) stimulated ET release time- and dose-dependently by up to 83.2% (P < .01) within 12 h in the absence of fetal calf serum and heparin. EPO induced an immediate significant rise in [Ca²⁺]_i from 58 ± 12 nmol/L to 495 ± 85 nmol/L (P < .01) with a subsequent slow return to 257 ± 3 nmol/L. During 2 h of incubation, the Ca-ionophore A 23187 (10⁻⁸ mol/L) moderately but significantly stimulated endothelial ET synthesis. However, the Ca-channel blocker verapamil, the intracellular Ca-release blocker TMB-8, and nickel, an unspecific calcium channel blocker, had no consistent effects on [Ca²⁺]_i or ET synthesis. The protein kinase C inhibitor H-7 stimulated basal [Ca²⁺]_i and cellular ET synthesis. The tyrosine kinase inhibitor genistein suppressed the EPO-induced rise in [Ca²⁺]_i and cellular ET synthesis. From these data we conclude that EPO may stimulate ET synthesis in vascular endothelial cells by activation of an EPO-receptor and via intracellular signalling mechanisms that comprise tyrosine kinase activation and a rise in [Ca²⁺]_i. Therefore, the systemic hypertensive effects of EPO may be due at least in part to local stimulation of vascular endothelial ET synthesis via calcium mobilization.     

Response of Human Insulinoma Cells to Extracellular Calcium Is Different from Normal B Cells

The preoperative determination of thelocalization of a small insulinoma is sometimesdifficult using routine imaging techniques. We have usedthe selective arterial calcium injection (SACI) test todetermine the location of the tumor preoperatively. Thepathophysiologic basis of the SACI test is based on theresponsiveness of insulinomas to calcium injected intothe feeding artery. In this study, we demonstrated the in vitro response of the insulinoma cellsto the extracellular calcium challenge by usingprimary-cultured insulinoma cells. Human insulinomacells were obtained from three patients. MIN6 cells(normal pancreatic B cells) were used as a control;their insulin response to various stimuli resembles thatof normal B cells. The insulin secretory dynamics inresponse to extracellular calcium were observed using a perfusion system. Second, the change ofthe concentration of cytosolic free calcium([Ca²⁺]_i) was monitored byfluorometry using fura-2/AM. When the concentration ofextracellular calcium ([Ca²⁺]_o) was changed from 2.54 mM to 10 mM, insulinsecretion from the insulinoma cells was markedlyincreased within 6 min (10- to 18-fold at maximum), andrapidly returned to the basal level; at the same time, [Ca²⁺]_i was immediatelyelevated and reached a peak within 1 min. In contrast,in the MIN6 cells, the insulin secretion and [Ca2+]iwere not significantly changed when[Ca²⁺]_o was switched to 10 mM. The results of these in vitro experiments agreedwith the clinical results of the SACI test. The positiveresponse of the insulinoma to the SACI test is probablydue to the different response of insulinoma cells to the extracellular calcium challengecompared with normal B cells. The role of[Ca²⁺]_i may be important in themechanism underlying the SACI test.     

3,4-Methylenedioxymethamphetamine (ecstasy)-induced hepatotoxicity: effect on cytosolic calcium signals in isolated hepatocytes

Abstract: Aims/Background: Hepatocellular damage has been reported as a consequence of 3,4-methylenedioxymethamphetamine (MDMA) intake. However, little is known about the cellular mechanisms involved. The present study was undertaken to evaluate the effects of MDMA on cell viability as well as free calcium levels ([Ca²⁺]_i) in short-term cultured hepatocytes. Reduced glutathione (GSH), adenosine-5′-triphosphate (ATP) and lipid peroxidation were investigated to evaluate the toxic effect of MDMA, in vitro, using freshly isolated rat hepatocytes. Methods: In order to measure cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i), rat hepatocytes were loaded with the Ca²⁺ indicator fura-2-acetoxymethylester (fura-2-AM). Results: A sustained rise of ([Ca²⁺]_i) after incubation with MDMA was the most noteworthy finding. In Ca²⁺-free medium, MDMA caused a reduced increase of ([Ca²⁺]_i). On the other hand, MDMA (0.1–5 mM) induced a concentration-dependent and time exposure-dependent GSH and ATP depletion. Although it did not reach statistical significance, GSH deficits were accompanied by a tendency to increase lipid peroxidation 3 h after MDMA incubation. Conclusions: The above data suggest that the marked rise of ([Ca²⁺]_i) and subsequent ATP and GSH depletion can lead to a rapid decrease in cell viability.     

Calcium-sensitizing inotropic agents in the treatment of heart failure: A critical view

Summary Interventions that augment the contractile state of the heart are associated with, or caused by, alterations in Ca²⁺ exchange in heart muscles. New inotropic agents have been developed that increase the sensitivity of the myofilaments to Ca²⁺. To examine the effect of calcium-sensitizing agents on force development, we measured systolic and diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) and constructed [Ca²⁺]_i-force relationships in normal (n=6) and myopathic human hearts (n=10). Using the bioluminescent calcium indicator aequorin, we found that the diastolic [Ca²⁺]_i was 225±52 nM in normal muscles, whereas in myopathic muscles diastolic [Ca²⁺]_i was significantly higher at 361±68 nM. Calcium-sensitizing agents that shift the [Ca²⁺]_i-force relationship toward lower [Ca²⁺]_i increase the diastolic force of myopathic hearts significantly more than in normal human hearts. This leads us to the conclusion that inotropic agents that increase the sensitivity of the myofilaments to Ca²⁺ further impair relaxation in myopathic hearts, resulting in a reduced contractile reserve and diminished actice force production.     

Thapsigargin-Insensitive Calcium Pools in Vascular Smooth Muscle Cells

Since sarcoplasmic Ca²⁺-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC)     

Insulin sensitivity increase after calcium supplementation and change in intraplatelet calcium and sodium–hydrogen exchange in hypertensive patients with Type 2 diabetes1

Aims/hypothesis To investigate the effect of oral calcium (Ca²⁺) supplementation on insulin sensitivity measured by the euglycaemic hyperinsulinaemic clamp, intraplatelet cationic concentration of Ca²⁺ ([Ca²⁺]_i) and the transmembrane sodium–hydrogen exchanger (NHE) activity in erythrocytes in subjects with Type 2 diabetes and hypertension. Patients and methods In this parallel randomized controlled single‐blinded trial, 31 patients were allocated to receive either 1500 mg of Ca²⁺ orally, daily (n = 15) or no treatment (n = 16) for 8 weeks. At baseline and at the end of the 8‐week period insulin sensitivity, [Ca²⁺]_i and the first isoform of NHE (NHE‐1) activity were measured. Results At the end of the study, subjects who received Ca²⁺ supplementation showed higher insulin sensitivity (ΔM‐value 0.32 ± 0.5 mmol/min P < 0.05) and lower [Ca²⁺]_i (125.0 ± 24.7 to 80.4 ± 10.6 nmol/l, P < 0.05, mean ± sem ) and NHE‐1 activity (79.5 ± 10.0 to 52.1 ± 6.4 mmol Na/l red cell/h, P < 0.05). None of the above parameters were changed in the control group. Simple regression analysis demonstrated the change in [Ca²⁺]_i significantly determined insulin sensitivity change (β = ?0.36, P < 0.05). Conclusions/interpretation Oral Ca²⁺ supplementation improves insulin sensitivity in patients with Type 2 diabetes and hypertension. These changes are likely to be mediated by changes in intracellular ionic Ca²⁺. NHE‐1 activity was also reduced after Ca²⁺ supplementation but its role in insulin sensitivity requires further investigation.     

Recruitment of individually (all-or-none) responding cells, rather than amplitude enhancement, is the single-cell mechanism subserving the dose-responsive activation of intracellular calcium second messenger signaling by the human lutinizing-hormone receptor

We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca²⁺]_i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH β- or α-subunits, evoked specific [Ca²⁺]_i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca²⁺]_i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca²⁺]_i responses were observed in 12–14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca²⁺]_i signal amplitude), and eliciting a [Ca²⁺]_i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca²⁺]_i spike-and-lateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn²⁺ following the [Ca²⁺]_i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca²⁺]_i resoonse, and pertussis toxin (PTX) did not alter LH-stimulated [Ca²⁺]_i signaling. However, overnight preincubation with LH reduced the percentage of [Ca²⁺]_i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca²⁺]_i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca²⁺ signaling response by recruiting an increasing number of [Ca²⁺]_i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca²⁺]_i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca²⁺]_i signal.     

Metformin Attenuates Agonist-Stimulated Calcium Transients in Vascular Smooth Muscle Cells

Metformin, an antidiabetic agent that increases insulin sensitivity, has been shown to lower blood pressure. However, the mechanism of action of metformin in vascular smooth muscle (VSM) cell is not fully understood. We have tested the hypothesis that metformin produces vascular changes by direct interaction with VSM cells by investigating its effect on platelet-derived growth factor (PDGF)- and angiotensin II (ANG II)-stimulated intracellular calcium concentration ([Ca²⁺]_i) and VSM cell proliferation in response to PDGF in cultured cells. VSM cells were cultured from rat thoracic aorta and [Ca²⁺]_i was estimated in single cells by image analysis. Treatment of VSM cells with 1 or 2 μg/ml metformin significantly decreased (p < 0.05) PDGF- or ANG II-stimulated [Ca²⁺]_i. Treatment of VSM cells with 1, 2, 5, or 10 μg/ml metformin had no significant effect on PDGF-stimulated [³H]-thymidine incorporation. However, metformin at pharmacological doses of 20 and 50 μg/ml significantly reduced (p< 0.05) PDGF-stimulated thymidine incorporation. We conclude that metformin mediates its vascular effects by attenuating agonist-stimulated [Ca²⁺]i.