Biomedical subjects

A Aviv

Publications and source records attributed to A Aviv.

At least 73 records · Page 4Linked to original sources

PubMed1993

K(+)-dependent Na+/Ca2+ exchange in human platelets.

Ca2+ influx via Na+/Ca2+ exchange was examined using fura-2 fluorescence techniques in human platelets loaded internally with Na+ by pretreatment with ouabain. In 140 mM LiCl, Ca2+ influx via the exchanger was completely dependent upon the presence of extracellular K+ (Km approximately 1 mM). In 140 mM N-methyl-D-glucamine (NMDG), Ca2+ influx was stimulated by K+ but was not absolutely dependent upon it; the inclusion of 20 mM NaCl in the NMDG medium restored the K+ dependence of Ca2+ influx. Stimulation of Ca2+ influx by K+ was confirmed by 45Ca2+ flux studies. 86Rb+ fluxes were measured to determine if Ca2+ and Rb+ were co-transported by the exchanger. The presence of extracellular Ca2+ stimulated 86Rb+ influx in ouabain-treated platelets in 160 mM LiCl or NMDG. The Rb+/Ca2+ influx ratio was 0.42 +/- 0.04 (n = 3) at [Rb+] = 0.2 mM and 0.89 at [Rb+] = 1.3 mM. Neither K(+)-dependent Ca2+ influx nor Ca(2+)-dependent Rb+ influx was observed in 160 mM NaCl or in platelets that had not been pretreated with ouabain, indicating that these fluxes resulted from Na+/Ca2+ exchange activity. Na(+)-dependent Ca2+ efflux was also shown to be dependent upon the presence of internal K+. These properties are similar to those of the Na+/Ca(2+)-K+ exchanger found in retinal rods, as distinct from the more widely distributed cardiac-type exchanger.

Blood Platelets↗

PubMed1993

Interaction between antidepressants and phosphoinositide signal transduction system in human platelets.

The present study was designed to evaluate the inhibitory effect of antidepressants on thrombin-induced phosphoinositide (PI) hydrolysis. Thrombin 5 units/ml induced a 100%-200% increase in platelet inositol phosphates (IPs) formation. This effect was inhibited in a dose-dependent manner by various heterocyclic antidepressants (IC50 40-170 mumol/L) The monoamine oxidase inhibitor, phenelzine, in concentrations up to 500 mumol/L, was devoid of inhibitory activity. The tricyclic antidepressants, (50 mumol/L) inhibited also thrombin-induced platelet aggregation by 32%-47%. No alteration in thrombin-induced IPs formation was detected in recovered major depressed patients (n = 15) maintained on clomipramine (75-150 mg/day). These results indicate that the heterocyclic antidepressants interfere with the thrombin-linked PI-signaling system. However, the inhibitory effect is achieved only in concentrations above the plasma concentrations obtained with therapeutic doses of these agents.

Antidepressive Agents↗

PubMed1993

Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: the roles of the Na+/Ca2+ exchange, the Na(+)-K(+)-2Cl- cotransport and cellular volume.

To explore further the mechanisms that regulate the Na+/H+ antiport in human platelets, we examined the effect of Na+ pump inhibition by ouabain and K+ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca2+ and Na+, coupled with an alkaline shift in the cytosolic pH set point for the Na+/H+ antiport. Inhibition of the Na+ pump by the removal of K+ from the medium increased the cytosolic Na+ but not the cytosolic Ca2+; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na+/H+ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na(+)-K(+)-2 Cl- cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na+ pump and the Na+/H+ antiport, mediated by the Na+/Ca2+ exchange and the cytosolic free Ca2+, and (b) a link between the Na+/H+ antiport and the Na(+)-K(+)-2Cl- cotransport through cellular volume.

Blood Platelets↗

PubMed1993

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains.

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the N-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗

PubMed1993

Thapsigargin-evoked changes in human platelet Ca2+, Na+, pH and membrane potential.

1. In this work we explored the effect of thapsigarin on the intracellular Ca2+, pH, Na+ and membrane potential in human platelets. These parameters were monitored using the fluorescent probes fura-2, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, sodium-binding benzofuran isophthalate, and 3,3'-dipropylthiadicarbocyanine iodide. 2. Thapsigargin caused an increase in the cytosolic Ca2+, coupled with cytosolic alkalinization. Thapsigargin-induced alkalinization was Na(+)-dependent, indicating that thapsigargin stimulated the Na(+)-H+ exchange. 3. Using Mn2+ as a Ca2+ surrogate, we showed that thapsigargin activated Ca2+ channels at relatively low levels of cytosolic Ca2+, suggesting that a rise in cytosolic free Ca2+ is not the signal for the activation of these channels. 4. Thapsigargin-induced increase in the cytosolic free Ca2+ was greater in Na(+)-containing medium than in Na(+)-free medium, suggesting that Na(+)-dependent mechanisms participate in the regulation of platelet cytosolic Ca2+. 5. Thapsigargin not only increased the cytosolic Ca2+, but also elevated the cytosolic free Na+. The latter effect was more pronounced in Ca(2+)-free medium, a finding that may indicate that some of the Na+ enters through Ca2+ entry pathways. 6. Finally, thapsigargin evoked sustained platelet hyperpolarization which was attenuated by charybdotoxin, indicating thapsigargin-induced stimulation of Ca(2+)-sensitive K+ channels. 7. Together these observations demonstrate a multifactorial effect of thapsigargin on platelets that can be utilized to further understand platelet ionic homeostasis.

Blood Platelets↗

PubMed1993

Kinetics of calcium transport across the lymphocyte plasma membrane.

We have investigated plasma membrane Ca2+ transport by monitoring the fluorescence of human peripheral T-lymphocytes loaded with fura 2. Thapsigargin (TG) was utilized the block the Ca(2+)-ATPase of the endoplasmic reticulum and elevate the cytosolic Ca2+ (Ca2+i). Ca2+ influx was inhibited by chelating extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The rate of decline in the Ca2+i signal of TG-treated lymphocytes after exposure to EGTA was used to assess Ca2+ extrusion across the plasma membrane. Initial rates of Ca2+i decline were examined in cells suspended in Na(+)-containing and Na(+)-free solutions; initial rates were linearly related to the [Ca2+]i at the onset of the Ca2+i decline and were unaffected by varying the extracellular Ca2+. Extracellular Na+ increased the rate of Ca2+ extrusion and decreased the threshold [Ca2+]i for extrusion, indicating a substantial role for the Na(+)-Ca2+ exchange in Ca2+i homeostasis. Both decreased temperature and calmodulin inhibition significantly slowed the Ca2+i decline in Na(+)-free HEPES-buffered solution, suggesting Ca2+ extrusion under these conditions was mediated by the Ca2+ pump. Protein kinase C (PKC) activation or inhibition did not affect the Ca2+i decline parameters. However, Ca2+ accumulation and Mn2+ (a Ca2+ surrogate) uptake were significantly and Mn2+ (a Ca2+ surrogate) uptake were significantly inhibited by activators of PKC. Cyclic nucleotides altered neither the parameters of the Ca2+i decline nor Mn2+ uptake. Thus human T-lymphocytes exhibit Na(+)- and Ca(2+)-dependent transporters characterized as the Na(+)-Ca2+ exchanger and Ca2+ pump. The main effect of PKC in these cells is the modulation of Ca2+ entry across the lymphocyte plasma membrane.

Biological Transport↗

PubMed1992

Characterization of Na(+)-K+ homeostasis of cultured human skin fibroblasts in the presence and absence of fetal bovine serum.

Previously, we demonstrated that removal of fetal bovine serum (FBS) from the medium of human skin fibroblasts resulted in an accelerated 86Rb+ washout, decreased cellular K+, and increased Na+ contents. In the present study we examined the mechanism underlying these changes. The efflux rate constant for 86Rb+, and the cellular contents of Na+ and K+ were measured. Verapamil (K1/2 = 15 microM) and chlorpromazine (K1/2 = 1 microM) reduced by approximately 70% the increased 86Rb+ washout evoked by FBS removal. The effect of the two drugs was additive at low, but not high, concentrations. Verapamil and chlorpromazine also attenuated the decrease in cellular K+ content and prevented the increase in cellular Na+ content associated with FBS depletion. Bumetanide (50 microM) was only partially effective in offsetting the enhanced 86Rb+ efflux and was completely without any effect on the cellular Na+ and K+ changes induced by FBS removal. In the presence of FBS, A-23187 produced a slight and transient increase of the 86Rb+ washout. The protein kinase C activator phorbol 12-myristate 13-acetate enhanced the 86Rb+ efflux in FBS-containing medium for a prolonged period but this increase was only a fraction of that caused by serum removal. Cellular Na+ and K+ contents were not changed by the phorbol ester. We conclude that FBS removal raises the cellular Na+ content, and enhances 86Rb+ efflux, through a calmodulin-dependent pathway activated by calcium influx.

Animals↗

PubMed1992

Effects of essential hypertension and antihypertensive medications on sweat formation.

OBJECTIVE: Sweat volume and ionic composition depend to a large extent upon the cytosolic free calcium level in secretory sweat cells and sodium and potassium transport in the reabsorptive sweat duct. Since essential hypertension and its treatment with antihypertensive drugs is likely to be associated with altered cellular ionic regulation, the objective of this research was to explore sweat formation and sweat parameters in hypertensive and normotensive subjects. DESIGN: Black and white hypertensive and normotensive subjects of both genders were studied. Essential hypertensives were on or off antihypertensive medication. METHODS: Pilocarpine iontophoresis was used to induce sweat in a 5-cm2 area of the middle forearm. Sweat was analyzed for volume, sodium and potassium concentrations. RESULTS: Females demonstrated lower sweat volumes after pilocarpine stimulation than males. Untreated hypertensive white males exhibited a higher pilocarpine-induced sweat volume and sweat sodium excretion than normotensive white males, whilst hypertensive white males on antihypertensive medication showed a lower sweat volume and sweat sodium excretion than both normotensive white males and untreated essential hypertensive white males. Although untreated hypertensive white females did not show significant alterations in sweat parameters, treated hypertensive white females exhibited lower sweat volume and sweat sodium excretion than both the normotensive and untreated essential hypertensive white females. These hypertension and drug related alterations were not present in hypertensive black males and females. CONCLUSIONS: The results are consistent with the heterogeneous nature of essential hypertension and the diversity of the response to antihypertensive therapy. They suggest that the effect of antihypertensive medication on sweat formation is mediated through cytosolic free calcium.

Adult↗

PubMed1992

Defects in membrane transport of ions as possible pathogenic factors in hypertension.

Although abnormalities in cellular ion transport have been shown in a variety of cells of essential hypertensives, the mechanistic link between these abnormalities and elevated blood pressure is poorly understood. Reduced sodium-potassium ATPase activity, with and without elevated levels of a circulating inhibitor of this transport system, has been reported by a number of studies. The recent characterization of the endogenous ouabain or its isomer will facilitate the testing of the hypothesis that salt-sensitive essential hypertension relates to higher levels of this factor. The erythrocyte sodium-lithium countertransport may serve as a marker for a subpopulation of essential hypertensives. However, this transport system has no physiologic counterpart and thus does not provide insight into mechanisms associated with altered cellular ionic homeostasis in essential hypertension. Increased activity of the sodium-hydrogen antiport in essential hypertension relates to an alkaline shift in the cytosolic pH set-point for activation of this transport system. This process may reflect increased cytosolic free calcium concentration with or without augmented protein kinase C activity.

Animals↗

PubMed1992

Endothelin-induced calcium responses in human vascular smooth muscle cells.

The effects of endothelin-1 (ET) on the cytosolic free Ca (Cai) and cytosolic pH (pHi) were examined in primary cultures of human umbilical artery (HUA) vascular smooth muscle cells (VSMCs), respectively, loaded with fura-2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In 1 mM Ca, ET produced a dose-dependent, biphasic increase in the signal with a maximal effect at 400 nM ET. At this concentration, ET produced a Cai transient (mean +/- SE; a rise from basal Cai of 86 +/- 16 to 216 +/- 33 nM) that lasted for approximately 50-60 s. The Cai transient was followed by a slow but sustained increase in Cai. Both ET-induced Cai transient and posttransient Cai were attenuated in Ca deficient medium or by verapamil and nicardipine. In contrast to ET, thrombin elicited only a monophasic Cai response in HUA VSMCs. This response was also partially sensitive to Ca removal or verapamil. KCl (45 mM) depolarization did not elicit a Cai response. However, the presence of voltage sensitive Ca channels in HUA VSMCs was demonstrated by enhanced Mn uptake in cells depolarized with KCl. Both ET and thrombin treatment did not alter pHi. HUA VSMCs demonstrated a single class of ET receptors (approximately 13,000 sites/cell) with an equilibrium dissociation constant of 0.34 nM. Nicardipine did not alter ET binding. These observations suggest a dual effect of ET on the Cai profile in HUA VSMCs that is mediated by Ca mobilization and Ca entry through Ca channels. The Ca entry could include influx through receptor-operated Ca channels, voltage-sensitive Ca channels, or both, but without a direct interaction between ET and these channels.

Calcium↗

PubMed1992

Platelet sodium-hydrogen antiport in obese and diabetic black women.

In this investigation we correlated platelet Na-H antiport parameters with blood pressure and serum lipids in a sample population of non-insulin-dependent diabetic obese, nondiabetic obese, and nondiabetic nonobese black women. Parameters of the Na-H antiport were examined in aspirin-treated platelets. These parameters were not altered in resting or in thrombin-stimulated platelets of diabetic patients. The activity index of platelet Na-H antiport after thrombin stimulation was positively correlated with the blood pressure (systolic blood pressure, r = 0.5320 and p = 0.0001; diastolic blood pressure, r = 0.5123 and p = 0.0017). Lower high density lipoprotein cholesterol levels were associated with an alkaline shift in the cytosolic pH set point for activation of the Na-H antiport. Highly significant correlations were also observed between the total cholesterol/high density lipoprotein cholesterol ratio and the cytosolic pH set point for activation of the Na-H antiport. These correlations were independent of diabetes or the body mass index. Together, these observations indicate that parameters of platelet Na-H antiport are altered with an increase in blood pressure and a decrease in serum high density lipoprotein cholesterol.

Adult↗

PubMed1992

Cyclic nucleotides attenuate thrombin-evoked alterations in parameters of platelet Na/H antiport. The role of cytosolic Ca.

In this work, we explored the role of cyclic nucleotides in modulating parameters of the Na/H antiport in human platelets. Sodium nitroprusside and iloprost, as well as cyclic nucleotide analogues, were used to raise cellular levels of cAMP and cGMP. Cyclic nucleotides reversed the thrombin-evoked alkaline shift in cytosolic pH set point and the activity of the Na/H antiport, concurrently with attenuation of thrombin-induced rise in cytosolic free Ca. No effect of cyclic nucleotides was observed in platelets not treated with thrombin, or platelets subjected to phorbol 12-myristate 13-acetate. cAMP did not reverse ionomycin-induced changes in the parameters of the Na/H antiport. Collectively, these observations indicate that cyclic nucleotides modulate the Na/H antiporter in human platelets through their effect on thrombin-evoked changes in cytosolic free Ca. Presumably, this effect holds for other agonists which stimulate phospholipase C, raise cytosolic-free Ca, and activate the Na/H antiport through protein kinase C dependent and protein kinase C-independent mechanisms.

Blood Platelets↗

PubMed1992

The roles of cell Ca2+, protein kinase C and the Na(+)-H+ antiport in the development of hypertension and insulin resistance.

There is evidence that the cytosolic free Ca2+, protein kinase C, and the Na(+)-H+ antiport cross-communicate with one another through positive and negative feedback mechanisms, thereby maintaining cellular Ca2+ and pH homeostasis. This triumvirate may play a role in the development of insulin resistance--a common characteristic of both essential hypertension and non-insulin-dependent diabetes mellitus. Circulating cells from patients with essential hypertension and non-insulin-dependent diabetes mellitus demonstrate elevated cytosolic free Ca2+, increased protein kinase C activity, or both, and these perturbations are associated with augmented activity of the Na(+)-H+ antiport. If present in other cells (e.g., striated muscle cells and adipocytes), these alterations could underlie insulin resistance in essential hypertension and non-insulin-dependent diabetes mellitus.

Animals↗

PubMed1991

Phorbol 12,13-dibutyrate binding to intact human platelets. The role of cytosolic free Ca2+.

The role of Ca2+ was examined in regulating the binding of phorbol 12,13-dibutyrate (PdBu) to intact human platelets. Alterations in the cytosolic free Ca2+ concn. [( Ca2+]i), but not extracellular Ca2+, substantially influenced the binding parameters of the phorbol ester. Ca(2+)-depleted platelets demonstrated a significant decline in the maximal binding capacity (Bmax), an increase in equilibrium dissociation constant (Kd) and a decrease in the Hill coefficient (h), suggesting the presence of Ca(2+)-sensitive and Ca(2+)-insensitive populations of PdBu-binding sites. In 1 mM-Ca2+ buffer, thrombin (0.1 NIH unit/ml) and ionomycin (0.5 microM) evoked a rise in [Ca2+]i to approx. 300-500 nM, associated with a significant decline in Kd, but without an apparent effect on Bmax. No effect of thrombin was observed on PdBu binding in Ca(2+)-depleted platelets. Inhibition of protein kinase C (PKC) by H7 was associated with a greater thrombin-evoked [Ca2+]i transient and a decline in Kd. Staurosporine also decreased the Kd for PdBu binding. We propose that this effect of the PKC inhibitors on the Kd was also [Ca2+]i-dependent. These observations in intact platelets indicate that the primary role of agonist- or non-agonist-induced rise in [Ca2+]i is to increase the affinity of PKC for PdBu and, presumably, endogenous diacylglycerol. However, in itself a rise in [Ca2+]i does not increase the Bmax, for PdBu binding.

Blood Platelets↗

PubMed1991

Combined resting-postural tremor of the head with a changing axis.

When essential tremor involves the head, it produces a rhythmic, periodic movement with a certain fixed frequency and a variable amplitude. The axis of the tremor has been described as either horizontal (no-no) or vertical (yes-yes) and is present when the head is in an action or postural position. Seven patients who differ in symptoms are described; one subgroup exhibited a combined resting-postural tremor of the head, sometimes with a changing axis; three patients had only postural head tremor with a changing axis.

Aged↗

PubMed1991

Behavioral profile and social competence in temporal lobe epilepsy of adolescence.

A group of 26 adolescents with temporal lobe epilepsy was compared with a matched control group of 26 adolescents with chronic bronchial asthma as well as with a group of 90 healthy adolescents, using the social competence measure and the behavior profile of the Child Behavior Checklist. Both chronically ill groups exhibited more social and behavioral problems than the healthy adolescents but were similar to each other on the social and behavioral profiles. The one area in which the temporal lobe epilepsy and asthmatic groups differed from one another was that of "schizoid" psychopathology in male subjects.

Adolescent↗

PubMed1991

Endothelin- and oxytocin-induced calcium signaling in cultured human myometrial cells.

The demonstration that endothelin (ET) induces rat uterine contraction, coupled with the observation that ET is present in human amniotic fluid, suggests that the myometrium may be an important target organ for this hormone. We show that in quiescent human myometrial cells ET produced a dose-dependent increase in cytosolic free Ca2+ (Cai2+), which was markedly attenuated when the cells were studied in Ca2(+)-free media. Preincubation with nicardipine, diltiazem, or verapamil reduced the ET-evoked Cai2+ transient by 30, 40, and 65%, respectively. The presence of voltage sensitive Ca2+ channels was demonstrated by Mn2+ quenching of fura-2. Activation of the Na+/H+ antiport could not be demonstrated with ET stimulation. In nonquiescent cells, the ET-evoked Cai2+ transient was significantly reduced, while the response to oxytocin was retained. This is at least partially explained by a reduction in Bmax (maximal binding capacity) for ET (mean +/- SEM) from 3,506 +/- 268 binding sites/cell in quiescent cells to 2,411 +/- 300 binding sites/cell, as well as 72% increase in Kd (equilibrium dissociation constant), in the nonquiescent cells. We conclude that, in human myometrial cells, ET and oxytocin modulate Cai2+ through independent receptors and propose that ET, like oxytocin, is an important endogenous modulator of uterine contractility.

Actins↗

PubMed1990

Agonist-evoked alkaline shift in the cytosolic pH set point for activation of Na+/H+ antiport in human platelets. The role of cytosolic Ca2+ and protein kinase C.

This work examines the roles of elevated cytosolic Ca2+ and stimulation of protein kinase C in agonist and non-agonist-mediated alkaline shift of the cytosolic pH set point for activation of Na+/H+ antiport in human platelets. Ca2(+)-depleted and control platelets were subjected to phorbol 12-myristate 13-acetate, thrombin, vasopressin, and ionomycin in 1 mM or Ca2+ free, nominally bicarbonate free media. To measure the cytosolic pH set point for Na+/H+ antiport activation, cells were acidified to different levels using the sodium propionate method. In some experiments protein kinase C was inhibited by staurosporine. Both protein kinase C stimulation and elevation of cytosolic Ca2+ can produce an alkaline shift in the pH set point for activation of Na+/H+ antiport in human platelets. However, the effect of Ca2+i on the pH set point predominates that of protein kinase C stimulation. Cytosolic Ca2+ is a prerequisite for agonist-evoked alkaline shift in the cytosolic pH set point for activation of Na+/H+ antiport. The cytosolic Ca2+ level is also essential for maintaining the basal cytosolic pH. These findings underscore the central role of cytosolic Ca2+ under basal and stimulated states in regulating cytosolic pH of human platelets.

Alkaloids↗