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S S Sheu

Publications and source records attributed to S S Sheu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Long-term clinical and morphological evaluation of primary membranoproliferative glomerulonephritis.

BACKGROUND: Membranoproliferative glomerulonephritis (MPGN) is a relatively rare primary glomerulonephritis (GN). Its incidence has decreased progressively in the past two decades. To improve knowledge of this rare GN, a retrospective review of 22 patients during a 12-year period was undertaken. METHODS: From November 1982 to December 1994, from a total 814 cases of primary GN, 22 patients with primary MPGN were diagnosed. Clinical data, medical records, renal pathology and outcome were reviewed. RESULTS: Patients included 15 male and 7 females, aged from 11 to 67 years. The average follow-up period was 46.3 months, with a range of 1 to 140 months. Tissue was available for electromicroscopic study in 11 cases; of which 9 cases fulfilled morphologic criteria of Type I MPGN; the other 2 cases were Type II MPGN. The clinical presentations at diagnosis included nephrotic syndrome (86.4%), impaired renal function (63.6%), microhematuria (50%), gross hematuria (31.8%) and hypertension (50%). Low serum C3 was found in 40.9% cases, 44.4% in Type I and 50% in Type III MPGN. The positive rate of hepatitis B virus infection was 22.7% with 33.3% in Type I and none in Type III MPGN. All 22 patients received various combined antihypertensive agents, immunosuppressant, anticoagulant and antiplatelet agents at diagnosis, but 17 had progressive disease, 4 maintained normal renal function with proteinuria and only 1 had complete remission. Fifteen patients, including six Type I and no Type III MPGN, progressed to end-stage renal failure. Both patients with Type III MPGN maintained normal renal function and responded to treatment. The 5 and 10 year actuarial renal survival rates were 33.3% and 16.7% respectively. The median kidney survival time was 51.2 months. CONCLUSIONS: A majority of cases with MPGN presenting with impaired renal function (86.7%) and hypertension (85%) at diagnosis progressed to end-stage renal failure. Delayed diagnosis and poor compliance were possible reasons for compared with for worse prognosis previous reports. But two patients with Type III MPGN had favorable prognosis previously described. Treatments generally failed to halt disease progression. Since at least five cases (22.7%) maintained normal renal function after treatments, a course of immunosuppressant is probably indicated if there is no contraindication. Further study with a larger population is warranted to clarify this issue.

Adolescent↗
PubMed1995

Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode.

A controversy in the field of bioenergetics has been whether mitochondria are capable of sequestering enough Ca2+ from cytosolic Ca2+ pulses to raise their intramitochondrial free Ca2+ level ([Ca2+]m). This is significant because an increase in [Ca2+]m has been linked to an increase in cellular metabolic rate through various mechanisms. To resolve this question, we exposed isolated liver mitochondria to physiological type pulses of Ca2+ produced using a pulse-generating system (Sparagna, G. C., Gunter, K. K., and Gunter, T. E. (1994) Anal. Biochem. 219, 96-103). We then measured the resulting mitochondrial Ca2+ uptake. The uniporter was previously thought to be the only specific Ca2+ uptake mechanism in mitochondria. Our studies have uncovered an additional uptake mechanism, the rapid mode of uptake or RaM, which functions at the beginning of each pulse and allows mitochondria to sequester a considerable amount of Ca2+ from short pulses. We have shown that the RaM is reset by decreasing the [Ca2+] between pulses for a very short time, making this uptake mode ideally suited for Ca2+ sequestration from Ca2+ pulse sequences. With rapid Ca2+ uptake occurring at the beginning of each pulse, liver mitochondria may be able to sequester sufficient Ca2+ from a short sequence of pulses to activate the cellular metabolic rate.

Animals↗
PubMed1995

Association of sorcin with the cardiac ryanodine receptor.

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A > 500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.

Animals↗
PubMed1995

Extracellular heparin inhibits Ca2+ transients and contraction in mammalian cardiac myocytes.

The effect of heparin on Ca2+ transients and cell shortening was studied in isolated cardiac myocytes from rat and guinea-pig ventricles. Ca2+ signals were measured with the fluorescent indicator fura-2. Heparin reversibly decreased Ca2+ transients and cell shortening in a dose-dependent manner. Half and complete blockade were obtained with 50microg/ml and 200microg/ml heparin, respectively. The dihydropyridine agonist BAY K 8644 (50nM) antagonized the effects of heparin. However, Ca2+ release elicited by caffeine (10mM) was not affected by heparin. The actions of heparin were also studied in multicellular preparations. In papillary muscle, heparin (5mg/ml) reversibly reduced the amplitude of the plateau of the action potential and the associated peak tension. BAY K 8644 (500nM) also antagonized these effects. It is proposed that heparin interacts with dihydropyridine-sensitive Ca2+ channels to cause a decrease of Ca2+ transients and contractility in heart.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Serum immunoglobulin E in primary IgA nephropathy.

The biosynthesis of human immunoglobulin E (IgE) is regulated by a complex network involving T and B lymphocytes. Diseases associated with high serum IgE (sIgE) levels are usually characterized by T cell disorders. Total sIgE level has been found to be of clinical relevance in minimal change nephrotic syndrome. However, the clinical significance has rarely been studied in primary IgA nephropathy (IgA N). We retrospectively studied 99 cases of primary IgA N. There were 59 males and 40 females with a mean age of 30.0 +/- 12.1 years. The mean follow-up duration was 45.9 +/- 31.1 months. Pathological grading was done according to the criteria of Meadow et al. Median sIgE for the entire group was 122.0 IU/ml (range: 2.8-5805 IU/ml) which was significantly higher than the healthy control group (median: 43,7 IU/ml, range: 5.0-1003 IU/ml, p < 0.001). However, when the IgA N cases were stratified into grades, only grade I (median: 514 IU/ml, range: 72.1-5805.0 IU/ml) and grade II (median: 229 IU/ml, range: 5.0-5464 IU/ml) patients had significantly higher sIgE than the control group (p < 0.0005 and p < 0.001 respectively). Patients with nephrotic ranged proteinuria (32 cases) were further classified into "stable" and "progressive" groups. The "stable" group had a significantly higher sIgE level (median: 922.0 IU/ml, range: 2.8-5805 IU/ml), compared to that of the "progressive" group (median: 55.3 IU/ml, range: 5.0-1600 IU/ml, p < 0.02). The effect of aggressive treatment (including corticosteroid and/or cyclophosphamide, cyclosporine) was also assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Autonomic modulation of action potential and tension in guinea pig papillary muscles.

The effects of alpha 1-adrenoceptor and muscarinic acetylcholine receptor stimulation on action potential and tension were studied in guinea pig papillary muscles obtained from both right and left ventricles. Stimulation of muscarinic acetylcholine receptors with carbachol produced a reduction of the action potential duration and a positive inotropic effect in papillary muscles from both ventricles. Both effects were concentration dependent and atropine sensitive. However, differential responsiveness was found upon alpha 1-adrenoceptor activation in muscles obtained from left and right ventricles. In right side papillary muscles, the alpha 1-adrenoceptor agonist, methoxamine, decreased the action potential duration and produced a positive inotropic effect. In contrast, methoxamine decreased the action potential duration but failed to produce a positive inotropic effect in left side papillary muscles. All methoxamine effects were antagonized by prazosin. Responses to maximum concentration of carbachol and methoxamine on the action potential duration and contractility were additive in right side papillary muscles. Phorbol 12,13-dibutyrate (PDB), a direct protein kinase C activator, also decreased the action potential duration in a manner that was additive to both carbachol and methoxamine. However, PDB reversed the positive inotropic effect of carbachol and methoxamine. The methoxamine-induced shortening of the action potential duration was prevented by pretreatment with indomethacin and nordihydroguaiaretic acid, blockers of arachidonic acid metabolism, but not by the protein kinase C antagonist, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7).(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Mitochondrial free Ca2+ concentration in living cells.

Evidence has accrued during the past two decades that mitochondrial Ca2+ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca2+ transport systems might be able to relay the changes of cytosolic Ca2+ concentration ([Ca2+]c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca2+ concentration ([Ca2+]m) become essential. In this article, we review the results from recent studies attempting to measure [Ca2+]m in living cells. In addition, the significance of each study is discussed.

Aequorin↗
PubMed1994

Differences in ligand binding and phosphoinositide turnover between M1 muscarinic receptor gene transfected cells and mouse and rat brain membranes.

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.

Adrenal Glands↗
PubMed1994

Mitochondrial calcium transport: physiological and pathological relevance.

Since the initiation of work on mitochondrial Ca2+ transport in the early 1960s, the relationship between experimental observations and physiological function has often seemed enigmatic. Why, for example, should an organelle dedicated to the crucial task of producing approximately 95% of the cell's ATP sequester Ca2+, sometimes in preference to phosphorylating ADP? Why should there be two separate efflux mechanisms, the Na+ independent and the Na+ dependent, both thought until recently to be driven exclusively either directly or indirectly by the energy of the pH gradient? Does intramitochondrial free Ca2+ concentration control metabolism? Is there evidence for any separate function of the mitochondrial Ca2+ transport mechanisms under pathological conditions? What is the relationship between mitochondrial Ca2+ transport, the mitochondrial membrane permeability transition, and irreversible cell damage under pathological conditions? First, we review what is known about control of metabolism, evidence for a role for intramitochondrial Ca2+ in control of metabolism, the cellular conditions under which mitochondria are exposed to Ca2+, characteristics of the mitochondrial Ca2+ transport mechanisms including the permeability transition, and evidence for and against mitochondrial Ca2+ uptake in vivo. Then the questions listed above and others are addressed from the perspective of the characteristics of the mechanisms of mitochondrial Ca2+ transport.

Animals↗
PubMed1994

Fabry's disease: clinical, pathologic and biochemical manifestations in two Chinese males.

Fabry's disease is a rare hereditary disease transmitted as an X-linked recessive trait with the primary metabolic defect of an enzyme alpha-galactosidase A, resulting in deposition of glycolipids (ceramide trihexoside) in various tissues, including the kidneys. Two sibling cases of Chinese adult male patients in a family with Fabry's disease were completely evaluated including the clinical, pathologic and biochemical studies. Both of the patients had the similar clinical manifestations such as telangiectases, proteinuria, acral pains, corneal opacities, tortuous renal vessels and recurrent fever. Chronic renal insufficiency was noted in Case 1, whereas Case 2 had normal renal function. Microscopic hematuria was noted in Case 1. In renal biopsy, LM showed foamy vacuolation of the glomerular visceral epithelial cells and EM showed widespread myelin bodies (Zebra bodies) in kidney tissues, most numerous in visceral epithelia in both cases. Those findings are diagnostic for Fabry's disease. The plasma activity of alpha-galactosidase of Case 1 was 0.8 and that of Case 2 was 1.0 (normal reference range: 8.5-18.5 nmol/hr/min). The plasma activity of alpha-galactosidase A of Case 1 was 0.4 and that of Case 2 was 0.8 (normal reference range: 7.9-16.9 nmol/hr/min). All the enzyme activities in both cases were much lower than those of normal subjects. In addition to clinical presentations, pathologic study and biochemical study with assays of plasma or serum activities of alpha-galactosidase and alpha-galactosidase A are important steps in the diagnosis of Fabry's disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The clinicopathological spectrum of renal amyloidosis.

BACKGROUND: Renal amyloidosis is an uncommon cause of nephrotic syndrome. The clinical conditions in Chinese people remain obscure. This is a retrospective review of the clinicopathological spectrum of 12 cases diagnosed in Taichung Veterans General Hospital from October 1982 to November 1993. METHODS: Charts and renal pathological slides were reviewed retrospectively. A scoring system was set to evaluate the degree of amyloid deposition in the renal tissue. The clinical profile, immunological data, pathological picture and final outcomes are presented and discussed, with literature review. RESULTS: There were 12 cases of primary amyloidosis including 1 case of multiple myeloma. All were confirmed by renal biopsy. The cases were all male with mean age of onset as 53.3 +/- 11.3 (range: 32 to 65 years). The mean follow-up duration was 22.9 +/- 32.8 months. The initial average creatinine clearance was 66.7 +/- 42.7 ml/min; mean daily urine protein was 7.0 +/- 4.1 grams. Nephrotic syndrome was the main clinical manifestation, present in all 12 cases. Other presenting symptoms and signs included: malaise in 7 cases; hypertension and anorexia in 4 cases; limb numbness in 3 cases; low back pain, dizziness and microhematuria in 2 cases each; anemia, headache, stroke, restrictive cardiomyopathy, hepatomegaly, syncope, body weight loss, dysphagia and skin itching in one case individually. Amyloid cardiomyopathy was present in 4 of the 8 patients who received echocardiography. The mean serum albumin level was 1.9 +/- 0.7 mg/dl, globulin level 3.1 +/- 1.1 mg/dl. Urinary Bence-Jones protein examination was performed in 8 cases; none revealed positive response. The mean immunoglobulin (Ig) level for the patients included: IgG 1018 +/- 901 mg/dl, IgA 262.0 +/- 313.8 mg/dl, IgM 104.8 +/- 84.2 mg/dl. There were at least 4 cases with high levels of one Ig but depressed levels in the others. M-component was shown by immunoelectrophoresis (IEP) in 90% of the cases. IEP impression in 10 cases revealed 2 cases of IgA lambda, 2 cases IgA kappa, 3 cases IgG lambda and 1 case IgG kappa monogammopathy, 1 case free lambda myeloma and 1 case negative. The ratio of kappa to lambda chain was 3:6. Bone marrow biopsy performed in 8 cases found only 1 case with multiple myeloma, one with amyloidosis; the other 6 cases were unremarkable. Mesangium was the site of heaviest amyloid deposition, followed by tubular basement membrane, artery and interstitium. The median survival time for those whose total score was lower than 3 points was 97.5 months; 3 to 5 points, 14 months; 6 points or more, 18.5 months. The median survival time was 21.6 months and 3-year-survival rate was 32.7%. The 2 cases with long-term survival were of 111 months and 84 months. The possible reason included: 1) Organ-limited renal amyloidosis; 2) Light amyloid deposition; 3) Younger age; 4) Other undetected favorable factors. CONCLUSIONS: 1) Renal amyloidosis is not a frequent diagnosis of nephrotic syndrome in Taiwan, but it should be suspected in every patient over 50 years old with a recent onset of proteinuria. 2) Renal amyloidosis can be diagnosed only by renal biopsy. 3) Primary renal amyloidosis is a disease of poor prognosis.

Adult↗
PubMed1993

Primary IgA nephropathy: a nine-year clinicopathologic study in the Veterans General Hospital-Taichung.

IgA nephropathy (IgA N) is the most common type of primary glomerulonephritis (GN) diagnosed in Taiwan. From February 1983 to May 1992, 194 patients with primary IgA N, representing 25.3% of the primary GN, were diagnosed by renal biopsy at this hospital. Clinicopathologic correlation was made in 175 cases of IgA N with adequate clinical and pathologic data including light-(LM), immunofluorescent (IF) and/or electron-(EM) microscopy. Modified classification of Meadow et al. was adopted for the histologic grading of glomerular lesions. Forty-nine biopsies (28.0%) showed Grade IV and V lesions (Grade IV, 10.9%; Grade V, 17.1%, respectively) in association with a high level of serum creatinine and a lower frequency of gross hematuria when compared with lesions of histologic Grades I to III. Patients with Grade V lesions revealed a high frequency of hypertension as compared with those with Grades I to IV. The frequencies of nephrotic range proteinuria in those with various grades of IgA N was not statistically significant in this study. One hundred and thirty patients were followed up for one to eight and half years or until end-stage renal disease (ESRD) developed (mean 3.9 years), excluding the biopsies done at ESRD or from the graft kidney. Forty-two patients (32.3%) had chronic renal insufficiency, of those 25 (19.2%) eventually developed ESRD. Seventy-five percent of the patients with histologic Grades IV and V showed progressive renal disease, while only 16% of patients with Grades I to III lesions revealed progressive disease, the latter indicating a more benign course (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Modulation of cytosolic free calcium concentration by alpha 1-adrenoceptors in rat atrial cells.

The effects of alpha 1-adrenoceptor stimulation by phenylephrine (PE) and beta-adrenoceptor stimulation by isoprenaline (ISO) on Ca2+ current (ICa) and free intracellular Ca2+ concentration ([Ca2+]i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of ICa, whereas large increases of peak ICa were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca2+]i and, initially, an increase in the height of peak [Ca2+]i transients. When the diastolic [Ca2+]i was increased to a greater extent, the amplitude of [Ca2+]i transients was decreased. Simultaneous measurements of [Ca2+]i and membrane potential showed that the increase in diastolic [Ca2+]i was associated with a depolarization of the membrane, and the greater amplitude of [Ca2+]i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca2+]i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca2+]i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca2+]i transients with only minor changes in diastolic [Ca2+]i. These results suggest that PE and ISO increase the amplitude of [Ca2+]i transients in rat atrium in different ways. The increase in [Ca2+]i transients in response to beta-adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca2+ channels causing a greater Ca2+ influx and a release of more Ca2+ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca2+]i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na(+)-Ca2+ exchange mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1992

Mechanism of extracellular ATP-induced increase of cytosolic Ca2+ concentration in isolated rat ventricular myocytes.

1. Changes in the cytosolic Ca2+ concentration ([Ca2+]i) of isolated rat ventricular myocytes in suspension were measured in response to extracellular ATP using the fluorescent Ca2+ indicators Quin-2 and Fura-2. 2. ATP produced a concentration-, time- and Mg(2+)-dependent, biphasic increase of [Ca2+]i whereas slowly hydrolysable ATP analogues produced a slow, monophasic increase of [Ca2+]i and the non-hydrolysable ATP analogues were without effect. 3. Extracellular Ca2+ was required for the ATP-induced increase of [Ca2+]i and pre-treatment of the cells with caffeine, ryanodine, verapamil or nimodipine partially inhibited the [Ca2+]i increase. 4. Whole-cell patch-clamp experiments revealed that ATP activated an ionic current that had a linear current-voltage relationship with a reversal potential near O mV. Quinidine, a putative P2 purinergic receptor blocker, abolished the ATP-activated current. The ATP-activated current was Mg2+ dependent. 5. Associated with the ATP-activated current was cellular depolarization. In a physiological solution, ATP depolarized cells to the threshold for the firing of action potentials. In the presence of the voltage-activated ion channel blockers tetrodotoxin, 4-aminopyridine, caesium and nitrendipine, ATP depolarized cells to -44 +/- 6 mV from a resting potential of -66 +/- 4 mV (n = 11). 6. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and autoradiography demonstrated that extracellular ATP stimulated the phosphorylation of several extracellular membrane-bound proteins. The phosphorylation of these proteins was concentration, time and Mg2+ dependent. Pre-treatment of cells with the slowly hydrolysable ATP analogues inhibited the ATP-induced phosphorylation. Adenosine 5'-O-3-thiotriphosphate (ATP gamma S) thiophosphorylated proteins with the same apparent molecular weight as the proteins phosphorylated by ATP. 7. These results suggest that the ATP-induced increase of [Ca2+]i is a result of the activation, possibly by protein phosphorylation, of a novel ion channel carrying inward current. The ATP-activated channel may be permeable to Na+ and Ca2+ and causes [Ca2+]i to rise. More importantly, this inward current depolarizes the cell to the threshold of inducing spontaneous firing of action potentials. The firing of action potentials results in the influx of Ca2+ through L-type Ca2+ channels which would trigger Ca2+ release from the sarcoplasmic reticulum and lead to the increase in [Ca2+]i.

Action Potentials↗
PubMed1992

Na+ efflux mechanisms in ventricular myocytes: measurement of [Na+]i with Na(+)-binding benzofuran isophthalate.

We characterized the Na(+)-sensitive dye benzofuran isophthalate (SBFI) with fluorescence microscopy in isolated, adult rat ventricular myocytes. When cells were loaded with SBFI by incubation with 10 microM of the acetoxymethyl ester, fluorescence excitation spectra were markedly attenuated below 340 nm and the isoemissive point was blue shifted by approximately 25 nm when compared with spectra from SBFI acid in buffered solutions. Fluorescence intensity (49 +/- 3%) was partially released by permeabilization of the sarcolemma with digitonin, suggesting that one-half of the dye molecules are sequestered subcellularly in a compartment shown most likely to be mitochondrial. Intracellular Na+ concentration ([Na+]i) was determined by in situ calibration using cation-selective ionophores and was found to be 14 +/- 2 mM in cells studied at 37 degrees C. The relative importance of Na+ efflux mechanisms in myocytes was investigated. Substitution of Ca2+ and Mg2+ with EGTA in the superfusing medium resulted in a reversible rise of [Na+]i from 13 +/- 2 to 31 +/- 5 mM, which was blocked by 1 microM verapamil. Cellular efflux of Na+ after loading in this manner was found to be insensitive to blockade of Na(+)-Ca2+ exchange but was abolished when Na(+)-K(+)-ATPase was inhibited with zero extracellular K+ concentration. We conclude that SBFI can be used to measure [Na+]i in ventricular cells nondestructively and without impalement. Na+ efflux after loading by Ca2+ and Mg2+ withdrawal is mediated by the Na+ pump with no measurable contribution from Na(+)-Ca2+ exchange.

Animals↗
PubMed1991

Ca2+ current and Ca2+ transients under action potential clamp in guinea pig ventricular myocytes.

Precise characterization of the magnitude and kinetics of transsarcolemmal Ca2+ influx during an action potential (AP) is essential for a complete understanding of excitation-contraction coupling in heart. Using a voltage-clamp protocol that simulated a physiological AP (AP clamp), we characterized the properties of the Ca2+ current (ICa) in guinea pig ventricular myocytes. The AP-generated ICa showed a complex time course that was different from ICa generated by a square pulse. ICa activated rapidly during the upstroke of the AP and then partially inactivated during the plateau. The fast component of ICa reached a peak value of -7.6 +/- 1.0 pA/pF at 2.40 +/- 0.30 ms after depolarization, followed by a slow component with a peak value of -2.9 +/- 0.4 pA/pF during the plateau. ICa generated by an AP was composed of both L- and T-type Ca2+ channels. T-type Ca2+ current contributed to the fast component of ICa and L-type Ca2+ current contributed to both fast and slow components of ICa. Activation of beta-adrenoceptors enhanced ICa with a maximal effect lasting throughout the entire plateau of the AP. Measurements of cytosolic Ca2+ transients using fura-2 indicated that the ICa was responsible for triggering Ca2+ release from the sarcoplasmic reticulum. The AP clamp provides a new approach for investigation of the relationship between ICa and Ca2+ transients under more physiological conditions.

Action Potentials↗
PubMed1990

Blockade of low and high threshold Ca2+ channels by diphenylbutylpiperidine antipsychotics linked to inhibition of prolactin gene expression.

The effects of diphenylbutylpiperidine (DPBP) antipsychotics on Ca2+ currents and prolactin (PRL) synthesis were studied in rat pituitary growth hormone (GH) cell lines (GH3 and GH4C1). In whole-cell patch-clamp experiments, DPBPs including fluspirilene, penfluridol, and pimozide at concentrations ranging from 0.25 to 5 microM each blocked current through low threshold T-type as well as high threshold L-type channels. Each of the drugs preferentially blocked T-type current, and complete inhibition was observed at concentrations as low as 1 microM. Inhibition of L-type channels by DPBPS was enhanced at depolarized holding potentials and promoted by prolonged channel activation. At concentrations similar to those which blocked Ca2+ currents, each of the three DPBPs markedly reduced basal PRL production by GH cells. PRL synthesis stimulated by the dihydropyridine Ca2+ agonist R5417 or thyrotropin releasing hormone (TRH) was also inhibited. The inhibitory effects of the DPBPs were observed at the level of gene transcription. Penfluridol and fluspirilene inhibited basal, Ca2(+)- and TRH-stimulated expression of a fusion gene construct containing the 5'-flanking sequence of the rat PRL gene linked to the luciferase gene. The effect was concentration-dependent with the IC50 values for both drugs of less than 1 microM. Nimodipine also reduced basal, R5417, and TRH-stimulated expression of the reporter gene construct. Similar results were obtained with a reporter gene construct containing the 5'-flanking sequence of the rat GH gene. The GH luciferase construct was only slightly responsive to R5417 and TRH; however, these responses were reduced by fluspirilene and nimodipine at concentrations of less than 1 microM. These studies demonstrate that the DPBP antipsychotics block T- as well as L-type Ca2+ channels in GH cells and inhibit PRL production at the level of transcription. They also indicate that functioning Ca2+ channels are necessary for TRH-stimulated PRL gene transcription.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗