Biomedical subjects

J L Ram

Publications and source records attributed to J L Ram.

At least 37 records · Page 2Linked to original sources

PubMed1996

Tamoxifen (estrogen antagonist) inhibits voltage-gated calcium current and contractility in vascular smooth muscle from rats.

Tamoxifen (Tx) has been used in breast cancer treatment and prophylaxis because of its antiestrogenic activity; however, Tx may also have beneficial cardiovascular effects and other actions mediated by mechanisms other than estrogen receptors. Previous studies showing interactions of Tx with Ca+(+)-channel blockers suggested that Tx may affect Ca++ channels, an hypothesis that was investigated using whole cell patch clamp techniques in vascular smooth muscle cells (cell line A7r5 and freshly dissociated cells) and by determining effects on contractions of isolated blood vessels. Tx reduced current through L-type Ca+2 channels, with an ID50 of 2 x 10(-6) M when applied by cumulative addition to A7r5 cells. With acute application, 10(-6) M Tx significantly reduced L-type current in A7r5 cells within 2 min to 88% of control (vehicle, 0.1% ethanol) in A7r5 cells, 67% of control in aortic vascular smooth muscle cells, and 60% of control in tail artery vascular smooth muscle cells. Tx also decreased the rate of inactivation of L-type current. Inhibition of T-type current by Tx was less than for L-type current but was significant at 10(-5) M Tx. Treatment of tail artery rings with Tx (10(-5) M, 15 min; 10(-6) M, 4 hr) reduced K+-elicited contractions. Since therapeutic concentrations of Tx during treatment may exceed 10(-6) M, these effects of Tx on vascular smooth muscle Ca++ channels and vessel contractions may have a role in the efficacy and side-effects of Tx treatment.

Animals↗

PubMed1995

Characterization of zebra mussel (Dreissena polymorpha) sperm morphology and their motility prior to and after spawning.

In order to understand more about the reproductive physiology of the zebra mussel (Dreissena polymorpha), we have examined the effect of spawning on the kinematics and fertilization ability of their spermatozoa. Sperm morphology was characterized using scanning electron microscopy, differences in sperm kinematics were examined using CASA following manual collection of sperm from the gonad or spawning induced by 5-hydroxytryptamine (5-HT), and the fertilization ability of spawned and manually removed spermatozoa were compared. D. polymorpha sperm have a tail 50 microns long, a midpiece consisting of four spherical mitochondria-rich regions approximately 0.6 micron in diameter, a conical nucleus 2.52 microns in length, and a cylindrical acrosome 0.72 micron in height and width. Kinematic analysis revealed that manually removed sperm exhibit significantly lower motion characteristics as compared to spawned sperm. Treatment with 5-HT had no significant effect on the sperm kinematics of either manually removed or spawned spermatozoa. These data suggest that D. polymorpha spermatozoa have a similar morphology to other bivalve sperm, spawning results in a significant enhancement in sperm kinematics, and 5-HT does not directly alter sperm motion.

Animals↗

PubMed1995

Effects of estradiol and progesterone on platelet calcium responses.

We investigated the effects of 17 beta-estradiol (beta E2), alpha-estradiol (alpha E2), and progesterone (P) on baseline and vasopressin (AVP)-induced [Ca2+]i in human platelets obtained from healthy male and female volunteers. Platelets were treated with beta E2, alpha E2, P, or ethanol vehicle for 30 min at 37 degrees C. In males, both beta E2 and P at 10(-5) mol/L reduced the AVP-induced rise in [Ca2+]i, to 72 +/- 3% (mean +/- SEM) and 53 +/- 3%, respectively. However, at 10(-6) mol/L only beta E2 had a significant effect (P < .02). In females, 10(-6) and 10(-5) beta E2 reduced the AVP response to 85.3 +/- 4.6% and 80.8 +/- 5.4% of control values, respectively. Progesterone (10(-6) and 10(-5) mol/L) reduced the AVP response to 83.8 +/- 5.1% and 60.3 +/- 2.0% of control values, respectively. The inactive estrogen alpha E2 had no effect on basal or AVP-induced rise in [Ca2+]i in either subject population, suggesting hormonal specificity. Neither beta E2 nor P affected baseline [Ca2+]i in either population. Thus, by attenuating [Ca2+]i responses in platelets, beta E2 and P may modulate platelet aggregation and atherosclerosis.

Adult↗

PubMed1994

In vivo and in vitro induction of germinal vesicle breakdown in a freshwater bivalve, the zebra mussel Dreissena polymorpha (Pallas).

Oocyte maturation and germinal vesicle breakdown (GVBD) was induced in zebra mussel (Dreissena polymorpha) oocytes by in vivo and in vitro application of serotonin (5-hydroxytryptamine, 5-HT), and in vitro application of 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, a 5-HT1A receptor agonist). Oocytes initiated GVBD approximately 30 minutes after exposure to 5-HT (10(-3) M) at 23 degrees C, and by 40-50 minutes after exposure, most oocytes lacked a germinal vesicle. An exposure time to 5-HT as brief as five to ten minutes was required to trigger the maturation process, which terminates in spawning of fertilizable oocytes in nearly all mussels. But, with an exposure time of less than five minutes, spawning was reduced by application of 10(-4) M methiothepin (a potent blocker of 5-HT-induced spawning in zebra mussels). Thus, the sequence of oocyte maturation events in zebra mussels was determined. Oocytes are arrested at the germinal vesicle stage (prophase I) within the ovary. 5-HT reinitiates the maturation process, including GVBD and spawning of metaphase I oocytes, which are further arrested until fertilization. To our knowledge this is the first demonstration of oocyte maturation induction by serotonergic ligands in a freshwater bivalve.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1994

Inhibition and sex specific induction of spawning by serotonergic ligands in the zebra mussel Dreissena polymorpha (Pallas).

Serotonin (5-hydroxytryptamine, 5-HT) stimulates spawning in the zebra mussel (Dreissena polymorpha), a macrofouling European bivalve that has recently invaded North America. To develop methods of controlling zebra mussel spawning, two vertebrate serotonin antagonists, methiothepin and metergoline, known to bind with high affinity to snail 5-HT receptors, were tested for their ability to block 5-HT-induced spawning in zebra mussels. Methiothepin inhibited 5-HT-induced spawning at concentrations as low as 10(-6) M. Metergoline (10(-4) M) inhibited 5-HT-induced spawning; however, at lower concentrations (10(-8) to 10(-5) M), metergoline by itself significantly induced spawning in male, but not female zebra mussels. Metergoline (10(-5) M)-induced male spawning was inhibited by 10(-5) M methiothepin. Thus, methiothepin is the most effective inhibitor and metergoline the most powerful inducer of spawning yet tested in zebra mussels.

Animals↗

PubMed1994

Ionic mechanisms mediating the acetylcholine-elicited contraction in Aplysia buccal muscle.

Ion channel mechanisms mediating acetylcholine (ACh)-elicited contractions in Aplysia buccal muscles were investigated using conventional force and membrane potential recording techniques and whole-cell voltage clamping of dissociated buccal muscle fibers. Previous work on buccal muscles of Aplysia showed that acetylcholine (ACh) depolarizes muscle fibers to, at most, -30 mV and that contraction is dependent upon extracellular calcium. In intact muscle, removal of either calcium or sodium from the extracellular medium reduced ACh-elicited depolarization. Absence of each ion reduced the depolarization approximately 50% at low [ACh], but at high [ACh], absence of sodium had a greater effect, while absence of calcium had a proportionally smaller effect on depolarization. Nifedipine (3 microM) reduced contraction by 50% with little accompanying change in depolarization. In whole cell voltage-clamping of isolated muscle fibers, ACh elicited an inward current at resting potential. The reversal potential for ACh-elicited current was -24 +/- 6 mV. The current-voltage curve was rectified, with a bend in the inward direction near -35 mV. In zero calcium medium, ACh-elicited current averaged 54% of control, and in zero sodium medium, 42% of control. Nifedipine reduced ACh-elicited current by less than 20%. Amiloride, an inhibitor of Aplysia buccal muscle contraction, reduced ACh-elicited current by 30-70%. A model based on the above results is that ACh depolarizes muscle fibers by opening channels with a reversal potential of about -30 mV that are permeable to calcium, sodium, and at least one other ion. Depolarization activates voltage-dependent calcium channels, enabling activator calcium to enter the fibers through both ACh-operated and voltage-dependent channels.

Acetylcholine↗

PubMed1994

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle.

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 +/- 0.1 (mean +/- SE, equivalent to 2 x 10(-5) M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10(-4) M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took > 40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT1A receptors and may be closely related to the gastropod 5-HTlym receptor.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1994

Effects of deionized water on sensitivity of zebra mussels (Dreissena polymorpha) to toxic chemicals.

Previous studies showed that zebra mussels begin to die within a few days of immersion in deionized water and that potassium was about 10 times more toxic to zebra mussels when applied in deionized than in normal aquarium water. This study investigated whether deionized water enhanced the sensitivity of zebra mussels to other toxic substances, by comparing viability of mussels after 24 hr exposure to various substances applied in aquarium water and deionized water. Toxicity of sodium hypochlorite was enhanced approximately two orders of magnitude in deionized water: in aquarium water, 10 mg/l < LD50 < 100 mg/l; in deionized water, 0.1 mg/l < LD50 < 1 mg/l. A shift of two orders of magnitude in toxicity was also observed for ouabain: in aquarium water, 10(-4) M < LD50 < 10(-3) M; in deionized water, 10(-6) M < LD50 < 10(-5) M. The toxicity of ouabain was unchanged by the addition of 37 mg KCl/l aquarium water. Virtually no shift in toxicity due to deionized water was observed for niclosamide and for Endod. For niclosamide, LD50 in deionized water was slightly less than 10(-5) mg/l and in aquarium water slightly above 10(-5) mg/l. For Endod, the LD50 was between 5 and 10 mg/l in both media. The largest shift in toxicity in deionized water occurred for Noxfish (rotenone), for which LD50 shifted from approximately 1 mg/l in aquarium water to less than 10(-4) mg/l in deionized water. Enhancement of toxicity due to deionized water is thus not an additive effect, but varies with the toxic substance, probably according to the mechanism of toxicity.

Animals↗

PubMed1994

In vitro relaxation of phenylephrine- and angiotensin II-contracted aortic rings by beta-estradiol.

In vivo studies suggest that 17 beta-estradiol (beta E) may regulate vascular tone. Results of recent studies suggest that beta E exerts rapid effects on intracellular calcium, possibly via cell surface receptors, distinct from conventional nuclear receptors for steroids. The present study was designed to determine whether beta E acutely modifies vascular smooth muscle contractile responses to phenylephrine (PE) and angiotensin II (AII). In experiments on tonic responses of aortic rings to 5 x 10(-8) mol/L PE, cumulative additions of beta E reduced tension at concentrations > 10(-6) mol/L. Contractile dose responses to PE were determined in rat aortic rings in absence of sex hormones and then after exposure to beta E (5 x 10(-6) mol/L, n = 6) or vehicle (ETOH, n = 6) for 30 min. beta E increased ED50 and reduced maximal responses. Application of 5 x 10(-6) mol/L beta E for 30 min also reduced the contractile response to 1 mmol/L AII from 69 +/- 4% (vehicle) to 47 +/- 6% (estradiol) of maximal KCl contraction (P < .025, n = 7). These data suggest that beta E acutely attenuates vasoconstrictor responses to PE as well as to AII, possibly by an effect exerted at the cell membrane level.

Angiotensin II↗

PubMed1994

17 beta-Estradiol attenuates voltage-dependent Ca2+ currents in A7r5 vascular smooth muscle cell line.

Previous studies have shown that 17 beta-estradiol (beta-E2) has a direct acute inhibitory effect on vascular smooth muscle (VSM) contraction. To investigate the mechanisms underlying this phenomenon, we utilized whole cell patch-clamping techniques to study effects of beta-E2 on voltage-dependent Ca2+ channels in cultured VSM cells (VSMC). T- and L-type Ca2+ currents were characterized with ramp and pulse protocols in A7r5 cultured VSMC. T-type current, inactivated in < 100 ms, was reduced by Ba2+ and was comparatively little affected by isradipine. L-type current required higher voltages to activate, inactivated slowly, was greatly increased by Ba2+, and could be completely inhibited by 5 microM isradipine. beta-E2 (10 microM) significantly reduced peak L-type Ba2+ current and T-type Ca2+ current within 1-2 min, whereas alpha E2 (a hormonally inactive isomer of estradiol) caused significantly less reduction in both types of current. Vehicle (0.1% ethanol) had no significant effect on either current. The inhibitory effect of beta-E2 on voltage-dependent Ca2+ currents may contribute to previously demonstrated beta-E2 attenuation of VSM contraction.

Animals↗

PubMed1994

Effects of pioglitazone on calcium channels in vascular smooth muscle.

Pioglitazone, an insulin-sensitizing, antidiabetic agent, has blood pressure-lowering effects in insulin-resistant hypertensive rats and attenuates growth factor-induced increases of intracellular Ca2+ in rat aortic vascular smooth muscle cells. To determine whether modulation of voltage-dependent Ca2+ channels plays a role in this association, we investigated the effects of pioglitazone on voltage-dependent current in cultured rat aortic (a7r5) and freshly dissociated rat tail artery vascular smooth muscle cells. Both cell types were studied with whole-cell patch-clamp techniques. Current through L-type Ca2+ channels was elicited with a voltage ramp in the presence of Ba2+ substituted for Ca2+. T-type Ca2+ current was studied using a two-pulse protocol that enabled the isolation of transient current. In a7r5 vascular smooth muscle cells, 2-minute application of pioglitazone (5 and 10 mumol/L) reduced L-type current by 7.9 +/- 1.0% (n = 8) (mean +/- SEM, number of cells) and 14.5 +/- 3.0% (n = 9) (P < .01, two-tailed paired t test), respectively. In contrast, 2-minute application of pioglitazone had no significant effect on T-type Ca2+ current. In freshly dissociated tail artery vascular smooth muscle cells, 2-minute application of 10 mumol/L pioglitazone had an insignificant effect (4.8 +/- 5.6% reduction); however, 25 mumol/L pioglitazone reduced L-type current by 27.3 +/- 7.2% (n = 5) (P < .01). Two-minute application of 0.1% or 0.2% dimethyl sulfoxide (vehicle) alone had no significant effects on currents in either type of vascular smooth muscle cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Spawning in the zebra mussel (Dreissena polymorpha): activation by internal or external application of serotonin.

The zebra mussel, Dreissena polymorpha, was recently introduced accidentally into the Great Lakes and, due in part to its prodigious reproductive capacity, is spreading rapidly in temperate fresh waters of North America. The present studies examine some of the mechanisms that regulate spawning in this animal. In August and September 1990 and in May 1991 injection of serotonin (5-hydroxytryptamine, 5-HT) induced ripe male, but not female, zebra mussels to spawn. During mid-summer 1991, 5-HT induced spawning in both males and females, and 5-HT could produce spawning responses by either injection or external application. External pH over a broad range (6.0 to 9.1) had no effect on spawning, neither inhibiting induction of spawning by 5-HT nor significantly eliciting spawning itself. With external application, 10(-3) M and 10(-4) M 5-HT caused spawning, but 10(-5) M and 10(-6) M did not. Cyproheptadine, a 5-HT receptor antagonist, reduced the response of both males and females by more than half. Spawning in response to 5-HT was blocked at 4 degrees C, but not at 12 degrees C, 20 degrees C, or 27 degrees C. For male zebra mussels morphological criteria for judging gonadal maturity were well-correlated with probability of spawning in response to 5-HT. For females, the likelihood of spawning in response to 5-HT was not tightly coupled to morphological maturity of the gonad, with many morphologically ripe females failing to spawn and some apparently immature animals releasing oocytes. Prior spawning reduced subsequent responsiveness and intensity of spawning of animals to 5-HT. These experiments support a role for 5-HT in regulating reproduction in zebra mussels and help define conditions by which zebra mussel spawning may be stimulated or inhibited.

Animals↗

PubMed1993

Effects of deionized water on viability of the zebra mussel, Dreissena polymorpha.

1. Zebra mussels exposed to deionized water (DW, changed daily) begin to die within several days. More than half the animals die in DW within a week. 2. Animals allowed to reattach themselves prior to exposure to DW live somewhat longer than detached animals exposed to DW but are still sensitive to the toxic effects of DW. 3. Small animals die in response to DW faster than large animals. 4. The toxic effect of DW can be suppressed by addition of 0.5 mM NaCl, 0.25 mM MgSO4, or small amounts of aquarium water. 5. KCl is about 10 times more toxic to zebra mussels when applied in DW than when applied in aquarium water. 6. The toxic effects of 37 mg KCl/l DW are not prevented by the addition of 0.5 mM NaCl or 0.25 mM MgSO4. 7. Stimulation of potassium transport mechanisms by DW may account for the increased toxicity of KCl in DW. Toxic effects of DW and increased toxicity of potassium in DW may have applications in controlling zebra mussel infestations.

Animals↗

PubMed1993

Hyperinsulinemia, insulin resistance, and hyperglycemia: contributing factors in the pathogenesis of hypertension and atherosclerosis.

Subtle abnormalities of carbohydrate metabolism and overt diabetes mellitus are both associated with a substantial increase in the prevalence of hypertension and the accelerated development of atherosclerosis. Hypertension is also a presumed independent risk factor for atherosclerosis, although some of the atherogenic properties of hypertension may be related to the recently recognized subtle metabolic abnormalities commonly found in persons with essential hypertension. The results of epidemiologic studies suggest that the elevated fasting and postprandial insulin levels that often occur in patients with essential hypertension, as well as in patients with type II diabetes mellitus, are an independent risk factor for atherosclerotic cardiovascular disease. Elevated glucose levels in patients with diabetes and hypertension appear to contribute to the acceleration of atherosclerosis, perhaps through toxic effects on the vascular endothelium. Other cardiovascular risk factors that are accentuated in persons with carbohydrate intolerance and hypertension include abnormalities in platelet function, clotting factors, the fibrinolytic system, and dyslipidemia. The goals of both nonpharmacologic and pharmacologic therapy for patients with abnormal carbohydrate metabolism and hypertension are to decrease cardiovascular risk as well as lower blood pressure.

Arteriosclerosis↗

PubMed1993

Regulation of Na+,K(+)-ATPase gene expression by insulin in vascular smooth muscle cells.

Na+,K(+)-ATPase gene expression and activity and its modulation by insulin were investigated in vascular smooth muscle cells (VSMC). Messenger RNAs encoding for alpha 1- and alpha 2-isoforms of Na+,K(+)-ATPase were identified in a pure population of cultured VSMC (a7r5 cells). The predominant isoform in VSMC was alpha 1, but only alpha 2 was regulated by insulin. Insulin treatment of VSMC for 1 and 6 h caused significant increases in mRNA encoding the alpha 2-subunit of Na+,K(+)-ATPase (n = 7, P < .01). Under similar conditions, insulin had no effect on alpha 1-Na+,K(+)-ATPase mRNA levels. VSMC treated with insulin exhibited a significant stimulation of ouabain inhibitable ATPase activity (1 h insulin, n = 3, v control, n = 3, P < .025; 6 h insulin, n = 3, v control, n = 3, P < .05). Changes in insulin regulation of Na+,K(+)-ATPase gene expression in vascular smooth muscle may be an important factor in the development of hypertension in diabetes.

Animals↗

PubMed1993

Insulin attenuation of vasopressin-induced calcium responses in arterial smooth muscle from Zucker rats.

Insulin attenuates agonist-induced vascular contractility of aortic rings and decreases vasopressin (AVP)-elicited increases in vascular smooth muscle cell (VSMC) intracellular calcium ([Ca2+]i). To determine if insulin's effects on AVP-induced [Ca2+]i responses are altered in an insulin-resistant and hypertensive state, we studied vascular smooth muscle calcium responses in VSMC derived from Zucker lean and obese rats. AVP concentration-response experiments revealed that VSMC derived from obese animals exhibited exaggerated [Ca2+]i responses over the range of 1 x 10(-10) to 1 x 10(-7) M AVP compared to lean controls (P < 0.05, by multiple analysis of variance). Insulin treatment (7 x 10(-7) M) decreased the [Ca2+]i response to 1 nM AVP by 66 +/- 8% and 71 +/- 9% in lean and obese VSMC, respectively. Similar decreases were observed with the 10 nM AVP stimulus (41 +/- 9% and 61 +/- 7%, for lean and obese, respectively). AVP receptor binding studies revealed that exaggerated [Ca2+]i responses in obese VSMC were not due to alterations in AVP-binding properties (no significant differences in ID50, Kd, or binding capacity in lean and obese VSMC preparations). In addition, insulin treatment (1 x 10(-7) M) resulted in no differences in AVP receptor-binding properties in either cell line. Therefore, exaggerated [Ca2+]i responses in obese VSMC are most likely due to a postreceptor abnormality. These abnormalities in VSMC [Ca2+]i metabolism preceed and may play a role in the development of hypertension in the Zucker obese rat. Although insulin resistance in Zucker obese rats has been demonstrated in several tissues, VSMC [Ca2+]i responses to AVP are, nonetheless, similarly attenuated by insulin in obese and lean VSMC preparations.

Animals↗

PubMed1992

Shock induces a long-lasting elevation of blood glucose in Aplysia.

Glucose, and not trehalose, was found to be the main blood sugar in Aplysia californica. Changes in blood glucose in response to stress produced by electric shock were measured in blood obtained both from animals dissected within ten minutes of shocking and from catheterized animals at various intervals, up to two and a half hours after the shock. Electric shock increased blood glucose levels. The rise in blood sugar continued as long as two and a half hours after shock.

Animals↗

PubMed1991

Voltage-dependent calcium current in dissociated smooth muscle cells of the buccal mass of Aplysia.

Isolated smooth muscle cells of the buccal mass of Aplysia contracted in response to depolarization elicited by a patch electrode in whole-cell configuration. With cesium-containing pipet solution and tetraethylammonium and 4-aminopyridine in the external solution depolarization elicited inward current. The voltage-dependent inward current was blocked completely by lanthanum (10 mmol.l-1), inhibited 80-90% by nifedipine (1 mumol.l-1), and was dependent upon extracellular calcium. These results showed that the voltage-dependent inward current was due to activation of voltage-dependent calcium channels (VDCaCH). Minimal depolarization to begin activating VDCaCH was -60 to -30 mV. Inward current peaked within 8 ms and then decreased rapidly to a lower level of relatively non-inactivating current. The initial peak current could be mostly inactivated by a depolarization to -20 mV for 500 ms. Nifedipine reduced both the peak current and the relatively non-inactivating current. Nifedipine inhibited high potassium-elicited contractions of both intact and dissociated muscle. These results suggested that VDCaCH mediates calcium influx which triggers contraction in molluscan smooth muscle fibers.

4-Aminopyridine↗