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P Haddad

Publications and source records attributed to P Haddad.

At least 37 records · Page 2Linked to original sources
PubMed1998

The SSRI discontinuation syndrome.

A characteristic selective serotonin reuptake inhibitor (SSRI) discontinuation syndrome appears to exist. It is usually mild, commences within 1 week of stopping treatment, resolves spontaneously within 3 weeks, and consists of diverse physical and psychological symptoms, the commonest being dizziness, nausea, lethargy and headache. SSRI reinstatement leads to resolution within 48 h. A transient stage of serotonin dysregulation appears central to causation with pharmacokinetic and pharmacodynamic differences accounting for the variation in incidence between the SSRIs. Discontinuation reactions are clinically relevant due to the associated morbidity, the potential for misdiagnosis and inappropriate treatment and because they may impair future antidepressant compliance. To minimize incidence, SSRIs, like other antidepressants, should be withdrawn gradually. Provisional diagnostic criteria for the SSRI discontinuation syndrome are proposed. Prospective studies are required to investigate the syndrome, particularly its effects on patient care.

Humans↗
PubMed1997

Effects of cold preservation and rewarming on rat liver cell volume regulation and concentrative amino acid uptake.

BACKGROUND & AIMS: Liver graft dysfunction is related to hypothermic preservation and may implicate altered cell volume homeostasis. The aim of this study was to evaluate how cold preservation and rewarming affect hepatocellular volume regulation and concentrative amino acid uptake. METHODS: Rat hepatocytes suspended for variable times in cold preservation solution were subsequently cultured at 37 degrees C. Cell viability was assessed by trypan blue exclusion. Cell volume was measured by videoplanimetric analysis of microscopic images. Amino acid uptake was assessed by measuring Na+-dependent cell swelling. RESULTS: Cell viability decreased as a function of cold storage time even as early as 4 hours. Upon hypotonic stress, cells swelled similarly irrespective of preservation time. However, the rate of subsequent regulatory volume decrease diminished significantly with increasing preservation time. Upon return to isotonic perfusate, cells shrunk to a similar extent, but the rate of regulatory volume increase was halved by cold storage beyond 24 hours. The rate of proline-induced cell swelling was diminished greatly after cold storage, unlike that induced by aminoisobutyric acid and glutamine. CONCLUSIONS: Hepatocellular volume regulatory mechanisms and concentrative proline uptake are significantly impaired after cold preservation and rewarming, unlike alpha-aminoisobutyric acid and glutamine uptake. This may favor chronic cell swelling after transplantation and participate in liver graft dysfunction.

Amino Acids↗
PubMed1997

Malignant and reactive cells from human lymphomas frequently express Fas ligand but display a different sensitivity to Fas-mediated apoptosis.

Fas ligand (FasL) is capable of inducing apoptosis of lymphoid cells by cross-linking with its natural receptor, Fas. We aimed to investigate the possible role of the Fas/FasL-mediated apoptosis in the development of human lymphomas. FasL mRNA was detected by reverse transcriptase-polymerase chain reaction in 38 out of 63 lymphoma biopsy specimens representative of various subtypes of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease. FasL was co-expressed with Fas mRNA in most cases. Flow cytometry (FACS) analysis showed a bright FasL staining in 31% to up to 75% of the total cell population from 14 out of 16 samples; the presence of the FasL protein was confirmed by Western blotting. Dual-color FACS analysis showed that FasL was expressed by T cells in B-NHLs and T-NHLs. A significant percentage of B cells in various B-NHLs also stained positively for FasL. Freshly separated neoplastic B cells from three FasL+ and one FasL- B-NHLs displayed a relative resistance to Fas-mediated apoptosis, when compared to reactive T cells isolated from the same tissue samples. In contrast, the sensitivity to Fas-mediated killing of the T cells isolated from two FasL+ T-NHLs was not uniform. These data show that (1) FasL is expressed in both neoplastic and reactive cells from a significant proportion of lymphoma cases, and (2) that the intratumoral FasL+/Fas+ reactive T cells are more sensitive to Fas-induced apoptosis than the neoplastic FasL+/Fas+ malignant B cells. A putative defect in the Fas/FasL pathway may thus favor the development of malignant B cell populations.

Apoptosis↗
PubMed1997

Different effect of cold storage and rewarming on three pH regulating transporters in isolated rat hepatocytes.

Disturbances in hepatic pH homeostasis are thought to participate in the functional damage to liver grafts caused by the cold preservation and warm reperfusion necessitated by transplantation surgery. We have used an in vitro model of isolated rat hepatocytes suspended in cold University of Wisconsin (UW) solution and subsequently cultured at 37 degrees C to evaluate liver cell pH regulatory mechanisms after cold preservation and rewarming. Cells were kept for up to 72 h in cold UW solution, and at 24-h intervals intracellular pH (pHi) was measured after 60-90 min of warm culture by cytofluorometry using the fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. When challenged with an alkaline load by isohydric HCO(3)(-)-CO(2) steps, hepatocytes exhibited similar maximal pH(i) values and recovered at the same rate, irrespective of cold storage time, indicating that Cl-/HCO(3)- exchange activity is quite resistant to hypothermic storage and subsequent rewarming. In parallel studies, cells were subjected to an acid load by the NH4Cl pulse technique in bicarbonate buffer containing 50 microM ethylisopropylamiloride to block Na+/H+ exchange. Despite similar nadir pH(i) (lowest pH(i) values due to acid load), the subsequent pH(i) recovery rate that reflects Na+-(HCO(3)-)n cotransport activity was increased significantly after hypothermic preservation. Hepatocytes were also perfused with a bicarbonate-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, and Na+/H+ exchange activity was evaluated using the same acid load protocol. Although cells always exhibited similar steady-state initial pH(i) and nadir, the rate of pH(i) recovery decreased significantly as a function of cold storage time in UW solution. Finally, intracellular buffering capacity was calculated from the sudden pH(i) changes induced by HCO(3)(-)-CO(2) steps or NH4Cl pulses and was found to remain stable throughout the 72 h of cold preservation. Therefore, the results strongly suggest that cold preservation and rewarming disturb hepatocellular pH regulatory mechanisms by attenuating Na+/H+ exchange and increasing Na+-(HCO(3)-)n cotransport, whereas Cl-/HCO(3)- exchange is not affected.

Adenosine↗
PubMed1997

Insulin induces Ca2+ influx into isolated rat hepatocyte couplets.

Isolated rat hepatocyte couplets were used to study the direct effect of insulin on intracellular Ca2+ homeostasis. Insulin induced a dose-dependent increase in hepatocellular Ca2+ that was gradual, generally monophasic, and reversible. Chelation of extracellular Ca2+ abolished the insulin-induced Ca2+ response, and this suppression was not related to an effect on insulin binding, as indicated by displacement studies. We thus tested the effect of several Ca2+ channel inhibitors on insulin-induced Ca2+ influx. Verapamil at 20 or 200 microM was without effect, whereas 500 microM nickel and 50 microM gadolinium strongly inhibited insulin-induced Ca2+ entry. Finally, we tested whether insulin-induced Ca2+ movements were implicated in the stimulation of mitogen-activated protein kinase (MAPK) activity, which we measured with the use of an immune-complex assay. Verapamil was without effect on the insulin-dependent stimulation of p44mapk activity, whereas addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, nickel, or gadolinium strongly inhibited the effect of the peptide hormone. Our results indicate that insulin triggers Ca2+ influx into hepatocytes, possibly through the opening of channels on the plasma membrane, and that this effect is important for insulin activation of MAPK.

Animals↗
PubMed1997

Serotonin reuptake inhibitor discontinuation syndrome: a hypothetical definition. Discontinuation Consensus panel.

Adverse events following discontinuation from serotonin reuptake inhibitors (SRIs) are being reported in the literature with increasing frequency; the frequency and severity of these symptoms appear to vary according to the half-life of the SRI, e.g., the incidence appears higher with the shorter half-life agents than with fluoxetine, which has an extended half-life. Yet, there have been no systematic studies of the phenomenon to date. Therefore, a group of experts convened in Phoenix, Arizona, to develop a clear description or definition of the phenomenon based on these reports. The SRI discontinuation syndrome, referred to as "withdrawal symptoms" in many anecdotal case reports, is distinctly different from the classic withdrawal syndrome associated with alcohol and barbiturates. Anti-depressants are not associated with dependence or drug-seeking behavior. SRI discontinuation symptoms tend to be short-lived and self-limiting, but can be troublesome. They may emerge when an SRI is abruptly discontinued, when doses are missed, and less frequently, during dosage reduction. In addition, the symptoms are not attributable to any other cause and can be reversed when the original agent is reinstituted, or one that is pharmacologically similar is substituted. SRI discontinuation symptoms, in most cases, may be minimized by slowly tapering antidepressant therapy, but there have been several case reports where symptoms occurred consistently even through repeated attempts to taper therapy. Physical symptoms include problems with balance, gastrointestinal and flu-like symptoms, and sensory and sleep disturbances. Psychological symptoms include anxiety and/or agitation, crying spells, and irritability. Further analyses of data bases and clinical studies are needed to define this proposed syndrome more clearly.

Depressive Disorder↗
PubMed1997

Newer antidepressants and the discontinuation syndrome.

Data on discontinuation phenomena associated with serotonin selective reuptake inhibitors (SSRIs) are derived primarily from (1) published case reports, (2) data bases of adverse drug reactions that have been spontaneously reported to national monitoring bureaus, and (3) clinical studies of drug discontinuation. Some of the symptoms seen on SSRI discontinuation, such as nausea, lethargy, insomnia, and headache, are similar to those reported with tricyclic discontinuation. However, SSRI discontinuation is also associated with novel symptom clusters, including problems with balance, sensory abnormalities, and possibly aggressive and impulsive behavior. Although generally mild and short-lived, discontinuation symptoms can be severe and chronic and have a major impact on the patient's lifestyle. The incidence of discontinuation symptoms varies widely among the different SSRIs; the highest rate is seen with paroxetine. The variation in incidence might be explained by the different pharmacokinetic and pharmacodynamic profiles of the SSRIs.

Adverse Drug Reaction Reporting Systems↗
PubMed1997

Possible biological mechanisms of the serotonin reuptake inhibitor discontinuation syndrome. Discontinuation Consensus Panel.

Although the number of documented serotonin reuptake inhibitor (SRI) discontinuation reactions is increasing, to date no systematic studies have been completed; therefore the mechanism of action for these reactions is not clearly understood. However, several hypotheses have been proposed. Researchers have postulated that discontinuation events result from a sudden decrease in the availability of synaptic serotonin in the face of down-regulated serotonin receptors. In addition, other neurotransmitters, such as dopamine, norepinephrine, or gamma-aminobutyric acid (GABA), may also be involved, although little research in this area has been published. Individual patient sensitivity, i.e., genetics or cognitive mindset, may also be a factor in SRI discontinuation phenomena. Finally, experts have hypothesized that since some symptoms associated with paroxetine withdrawal are similar to those of tricyclic antidepressant discontinuation, they may be caused by cholinergic rebound.

Down-Regulation↗
PubMed1997

Dexfenfluramine modulates hepatic glycogen metabolism by a calcium-dependent pathway.

In this study, the mechanism of action of dexfenfluramine (DEXF) at the hepatic level was investigated. The drug is shown to bind to the alpha 1-adrenergic receptor and to increase intracellular calcium in isolated rat hepatocytes, thereby activating phosphorylase via a calcium-dependent mechanism. Moreover, phosphorylase activation by DEXF was inhibited by different agents that interfere with the alpha 1-adrenergic signalling system: prazosin, phorbol 12 alpha-myristate 13 beta-acetate (PMA), and DEXF itself. We also show that phosphorylase activation induced by catecholamines and analogues (epinephrine, phenylephrine), whose actions are mediated by a calcium-dependent mechanism, was counteracted by the drug in the submillimolar range (0.1-1 mM). The activation of glycogenolysis by the drug is accompanied by a stimulation of the glycolytic flux (54% increase in lactate plus pyruvate accumulation), consistent with an increase in fructose-2,6-bisphosphate (F-2,6-BP) levels (36%). These results indicate that the interaction of DEXF with the alpha 1-adrenergic receptor channels glucose 6-phosphate derived from glycogen away from glucose production into the glycolytic pathway.

Adrenergic Agonists↗
PubMed1995

In vitro activity of S 9788 on a multidrug-resistant leukemic cell line and on normal hematopoietic cells-reversal of multidrug resistance by sera from phase I-treated patients.

The triazinoaminopiperidine derivative S 9788 is a new multidrug-resistance modulator that is currently being evaluated in phase I clinical trials. In this study, the reversal effect of S 9788 in comparison with verapamil was shown in vitro in human T-leukemic CCRF-CEM/VLB cells expressing the multidrug-resistance (MDR) phenotype. S 9788 increased in a dose-dependent manner the cytotoxic activity of doxorubicin or vinblastine, with complete reversal of resistance occurring at 2 microM for a concomitant continuous exposure (96 h) to the cytotoxic drugs. At respective concentrations equivalent to the IC10 value (the concentration inhibiting 10% of cell growth), S 9788 was 44 times more potent than verapamil in CCRF-CEM/VLB cells. S 9788 at 2 microM did not enhance the in vitro toxicity of doxorubicin or vinblastine in the human normal bone-marrow erythroid (BFU-E) and myeloid (CFU-GM) progenitors. The effect of exposure duration and concentrations on the synergistic action of modulator and cytotoxic agent closely depended on the cytotoxic agent studied. Post-incubations with S 9788 alone after a 1-h coadministration with vinblastine and S 9788 dramatically increased the reversal effect (4-41 times) in proportion to both the duration of postincubation and the concentration of S 9788. In contrast, for doxorubicin resistance, post-incubation with S 9788 alone induced a maximal 2-fold increase in the reversal effect that was not proportional to the post-incubation duration. In patients treated with S 9788 as a 30-min intravenous infusion during phase I trials, a good correlation was found between the serum levels of S 9788 and the ability to reverse MDR in CCRF-CEM/VLB cells. The reversal effect was dose-dependent and was effective beginning at a plasma concentration of 0.25 microM. These data form a basis for the design of phase II trials using a combination of a loading dose of S 9788 given before vinblastine or doxorubicin administration followed by a maintenance infusion of S 9788 alone for a period of 2-24 h.

Antineoplastic Agents↗
PubMed1995

The impact of cancer on key relatives: a comparison of relative and patient concerns.

This study examines the impact of diagnosis on key relatives of 108 newly diagnosed cancer patients. Cancers of the breast, colon, female reproductive tract and testicle were included. Relatives completed a self-report checklist of concerns and the General Health Questionnaire (GHQ) (28 items). Patients were administered a corresponding concerns checklist as part of a semi-structured interview. More relatives than patients expressed each of the nine common concerns; the discrepancy was greatest for concern about the patient's emotional reaction, reported by 95% of relatives and only 18% of patients. The total relatives' concern score (median 16.0) was significantly greater than the patients' score (median 4.0) (P < 0.0001). Cancer type, age of patient and nature of relationship to patient had little impact on the frequency of relatives' concerns. 52 (48%) relatives scored above the usual cut-off on the GHQ, suggesting significant psychological morbidity. Relatives of newly diagnosed cancer patients report high levels of concerns and psychological distress, and deserve greater attention than they currently receive.

Adolescent↗
PubMed1995

Natriuretic peptides inhibit nicotine-induced whole-cell currents and catecholamine secretion in bovine chromaffin cells: evidence for the involvement of the atrial natriuretic factor R2 receptors.

There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R1C but not the ANF-R1A receptor subtype. Evidence for the existence of ANF-R2 receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R2-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R2 receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R2 receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that the effect of CNP is predominantly mediated by the ANF-R2 and not the ANF-R1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCl-induced secretions were affected by natriuretic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1995

Involvement of a tyrosine kinase pathway in the growth-promoting effects of angiotensin II on aortic smooth muscle cells.

Angiotensin II (AII) is a growth factor that stimulates protein synthesis and induces cellular hypertrophy in aortic smooth muscle cells (SMC). This trophic effect is mediated by the AT1 subtype of AII receptors. However, very little is known about the cellular signaling pathways involved in this response. In the present study, we examined the role of protein tyrosine phosphorylation in the growth-promoting effects of AII on rat aortic SMC. The addition of AII to quiescent aortic SMC induced tyrosine phosphorylation of multiple substrates, as revealed by antiphosphotyrosine immunoblotting. This response was blocked by preincubation with the AT1-selective antagonist losartan. To explore the functional role of this signaling pathway, we performed experiments with two mechanistically distinct tyrosine kinase inhibitors. Treatment of quiescent aortic SMC with genistein and herbimycin A abolished the stimulatory effect of AII on overall protein tyrosine phosphorylation. Similarly, the two inhibitors prevented AII-induced tyrosine phosphorylation of the cytoskeletal protein paxillin. Under the same conditions, incubation with genistein or herbimycin A did not interfere with AII binding to the AT1 receptor and did not significantly affect AII-stimulated inositol-1,4,5-trisphosphate production and Ca2+ mobilization. In parallel to their selective action on tyrosine phosphorylation, both genistein and herbimycin A completely inhibited AII-stimulated protein synthesis in a dose-dependent manner. In contrast, the two inhibitors were much less potent in preventing the trophic effect of phorbol-12-myristate 13-acetate in these cells. We further demonstrate that genistein and herbimycin A did not prevent mitogen-activated protein kinase activation and c-fos gene induction, which is consistent with the notion that these downstream effectors do not link AII-induced tyrosine phosphorylation to protein synthesis. These results provide evidence that tyrosine phosphorylation has a critical role in cellular hypertrophy and is involved in AII action in vascular SMC.

Angiotensin II↗
PubMed1995

Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes.

Extreme variations in extracellular Ca2+ concentrations ([Ca2+]e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca2+]e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca2+]e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca2+]e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca2+]e prevailing in vivo. Intracellular Ca2+ concentrations ([Ca2+]i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca2+]e strongly influenced the [Ca2+]i response to EGF with significantly smaller [Ca2+]i increases in hepatocytes of normal rats kept in low [Ca2+]e compared with those kept in normal [Ca2+]e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2+]e. In normal [Ca2+]e, the EGF-induced increases in [Ca2+]i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca2+]e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca2+]e. Furthermore, the blunted responses in [Ca2+]i mobilization and DNA synthesis associated with hypocalcemia could not be overcome by increasing concentrations of EGF nor by normalization of [Ca2+]e in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Product of the steel locus suppresses apoptosis in hemopoietic cells. Comparison with pathways activated by granulocyte macrophage colony-stimulating factor.

Steel factor (SF), also referred to as Kit ligand, stem cell factor, or mast cell growth factor, is essential for the development of hematopoietic stem cells in vivo. It is shown here that SF is mainly a survival factor for hemopoietic cells with little if any proliferative effect. In contrast, granulocyte macrophage colony-stimulating factor (GM-CSF) acts both as a survival factor and as a potent growth factor. We have probed the pathways activated by SF and GM-CSF in suppression of active cell death (apoptosis) using two classes of inhibitors: Tyrphostins that are specific inhibitors of protein tyrosine kinase, and amiloride derivatives (5-(N,N-ethyl-n-isopropyl)amiloride and 5-(N,N-hexamethylene)amiloride) that have been designed as specific inhibitors of the Na+/H+ antiporter. Both SF-dependent and GM-CSF-dependent pathways are sensitive to inhibition by Tyrphostins with, nonetheless, a quantitative difference. All Tyrphostins tested are more potent inhibitors of c-Kit than of GM-CSF receptor triggered pathways, the most striking being Tyrphostin B42 that is 10 times more potent. In contrast to the discrepancy in Tyrphostin dose-response curves, titration curves for 5-(N-ethyl-n-isopropyl)amiloride and 5-(N,N-hexamethylene)amiloride are comparable in SF- or GM-CSF-stimulated cells. Furthermore, SF induces a rapid and sustained alkalinization of the intracellular pH, as assessed with the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5-carboxyfluorescein. Taken together, our data indicate that input from two distinct pathways with discrepancy in immediate early events, that of c-Kit and GM-CSF receptor, results in a common output, activation of the Na+/H+ antiporter and suppression of apoptosis by the two ligands.

Alkaloids↗
PubMed1994

Different modulation of hepatocellular Na+/H+ exchange activity by insulin and EGF.

Indirect evidence suggests that insulin, like epidermal growth factor (EGF), stimulates liver cell Na+/H+ exchange. We directly studied the effect of insulin on intracellular pH (pHi) and the Na+/H+ exchanger in isolated rat hepatocytes with the fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The effects of insulin were compared with those of EGF. Studies were carried out in the absence of HCO3- and in the presence of acetazolamide to isolate the Na+/H+ exchanger from other pH regulatory mechanisms. Insulin (9 nM) caused a reversible acidification of baseline pHi, whereas no significant effect was observed with EGF (30 nM). pHi was acidified by two different methods (NH4Cl pulse and external Na+ removal) to assess liver cell Na+/H+ exchange activity. In the NH4Cl pulse experiments, insulin had no significant effect on the Na+/H+ exchanger compared with the control (0.141 +/- 0.009 pH units/min, n = 14, and 0.122 +/- 0.023 pH units/min, n = 16, respectively). However, in the same conditions, EGF nearly doubled the rate of the Na+/H+ exchange activity (0.193 +/- 0.015 pH units/min, P < 0.05, n = 6). In the Na+ removal experiments, EGF again significantly increased the pHi recovery rate (0.542 +/- 0.032 pH units/min, n = 3) compared with the control (0.227 +/- 0.028 pH units/min, n = 5) and insulin (0.245 +/- 0.053 pH units/min, n = 5). Compared with control conditions, a subchronic administration of insulin (9 nM) in vitro had no significant effect on the Na+/H+ exchanger, nor did it affect baseline pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Chloride↗
PubMed1994

Chronic hypocalcemia of vitamin D deficiency leads to lower intracellular calcium concentrations in rat hepatocytes.

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Animals↗