Effects of propranolol on phosphatidate phosphohydrolase and mitogen-activated protein kinase activities in A7r5 vascular smooth muscle cells.

High doses of propranolol inhibit phosphatidate phosphohydrolase (PAP) activity in intact cells, thus blocking metabolism of phosphatidic acid (PA), product of the phospholipase D (PLD) reaction. Vasopressin and phorbol ester activate PLD and ERK (extracellular signal-regulated protein kinase) mitogen-activated protein kinases in A7r5, a rat vascular smooth muscle cell line. Propranolol increased PA levels in intact A7r5 cells and inhibited cytosolic PAP and membrane calcium-independent phospholipase A2 but did not activate PLD or enhance agonist-induced PA accumulation. Incubation of cells with 200 microM propranolol for 10-45 min markedly elevated PA but caused only partial activation of ERKs. Propranolol and other lipophilic amines caused a time- and dose-dependent detachment of cells from their substrate. These results confirm that elevation of PA is not a strong signal for ERK activation and emphasize that caution should be exercised in using propranolol as a PAP inhibitor in intact cells.

Reduced glucose transport in sperm from roosters (Gallus domesticus) with heritable subfertility.

Roosters homozygous for the rose comb allele (R/R) are subfertile. In previous research, these subfertile roosters were characterized by an in vitro sperm penetration assay as having limited sperm motility. The objectives in the present study were to characterize sperm motility by computer-assisted sperm motion analysis and to account for a mechanism underlying poor sperm motility. Percentages of motile sperm differed between subfertile males and fertile controls (r/r) by 29% (p < 0.001). The concentration of intracellular ATP in sperm form subfertile roosters was less than in that from fertile controls (p < 0.001). The genotypic difference is sperm motility, as measured with the sperm penetration assay, was maintained when ATP production was dependent on anaerobic glycolysis (p < 0.001). In this case, sperm were incubated with exogenous glucose and cyanide. Consequently, we could not attribute the genotypic difference in sperm mobility to mitochondrial respiration. In contrast, glucose transport, as measured by the uptake of [1,2-3H]-2-deoxy-D-glucose, was reduced in sperm from subfertile roosters (p < 0.001). Neither hexokinase nor glyceraldehyde-3-phosphate dehydrogenase activity differed between genotypes (p > 0.05). Likewise, lactate dehydrogenase activity did not differ between genotypes (p > 0.05). As evidenced by creatine kinase activity and dynein ATPase activity, neither the potential for energy transfer nor utilization within the axoneme differed between genotypes (p > 0.05). Therefore, we attribute the subfertility of roosters homozygous for the rose comb allele to decreased spermatozoal glucose transport.

Extracellular signal-regulated kinase-1 and -2 respond differently to mitogenic and differentiative signaling pathways in myoblasts.

In this report we show that extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) respond differently to signals that elicit proliferation and/or differentiation of myoblasts using the C2C12 cell line and nondifferentiating mutant NFB4 cells derived from them. Induction of differentiation by withdrawal of serum rendered ERKs in C2C12 myoblasts relatively insensitive to restimulation by serum. Instead, myogenic differentiation of C2C12 cells was associated with sustained activation of ERK-2 dependent on the insulin-like growth factor II (IGF-II) autocrine loop. By contrast, mutant NFB4 cells cultured under the same conditions remained proliferative and demonstrated robust activation of ERKs in response to serum. Similarly, a Gi-dependent signaling pathway induced activation of ERKs in NFB4 cells, but not in C2C12 cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells partially rescued by prolonged IGF-I treatment, ERK activity remained responsive to Gi-dependent LPA stimulation, whereas rescue of NFB4 cells by constitutive expression of myogenin or MyoD, associated with activation of the IGF-II autocrine loop, rendered the Gi-signaling pathway refractory to LPA stimulation. Relatively high levels of G(alpha i2) were detected in NFB4 cells and IGF-I treated NFB4 cells, which correlated with responsive Gi signaling. Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or treatment with IGF-II was associated with loss of G(alpha i2) and inhibition of Gi-dependent signaling. Thus, IGF-I and IGF-II activate distinct signaling cascades, with IGF-II eliciting a stronger differentiation effect correlated with down-regulation of G(alpha i2) protein. Short-term stimulation of NFB4 cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1 and -2 activation. Transient stimulation of NFB4 cells with IGF-I while blocking activation of Gi-proteins is with pertussis toxin resulted in preferential activation of ERK-2 characteristic of differentiated C2C12 cells, suggesting that proliferation induced by IGF-I is Gi-dependent and separable from the IGF-I-signaling pathway that leads to differentiation.

Inhibition of cyclic AMP accumulation by endothelin is pertussis toxin sensitive and calcium independent in isolated adult feline cardiac myocytes.

The aims of this study were to determine whether endothelin-1 (ET-1), a positive inotropic agent, altered the production of cyclic AMP (cAMP) in adult feline cardiac myocytes and to characterize the effect with respect to G-protein-coupling and calcium regulation of adenylyl cyclase. ET-1 inhibited basal and/or stimulated cAMP accumulation in the intact cardiac myocyte and in membrane preparations in a dose-dependent manner. In intact cells, maximal inhibition of forskolin-stimulated cAMP accumulation was 90-95% with an EC50 of 5 x 10(-10) M. Inhibition of isoproterenol-stimulated cAMP was biphasic with maximal inhibition of 70% observed by 10(-11)M; at higher doses inhibition was not consistently observed. The inhibitory response to ET-1 occurred in the absence or presence of isobutylmethylxanthine suggesting that activation of cAMP phosphodiesterases was not the means for reducing cAMP levels. Prior exposure of cardiac myocytes to 100ng/ml pertussis toxin blocked the inhibitory action of ET-1, indicating that this response is mediated through the involvement of a pertussis toxin-sensitive G-protein such as Gi. Studies carried out in the absence of extracellular calcium and under conditions of cell-loading with the intracellular calcium chelator, 1,2-bis-(2-aminophenoxy)-ethane-N,N,N'N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM), suggest that the mechanism by which ET-1 inhibits cAMP accumulation is not calcium-dependent. Thus, inhibition of cAMP accumulation by ET-1 appears to be mediated through a pertussis toxin sensitive protein rather than by activation of phosphodiesterases or calcium inhibition of cardiac forms of adenylyl cyclase. Though unlikely to play a role in the positive inotropic effect of ET-1, transduction of ET-1 responses through Gi suggests another means for regulation of growth in these adult cardiac myocytes.

Effects of endothelin on mitogen-activated protein kinase activity and protein synthesis in isolated adult feline cardiac myocytes.

The growth-promoting effects of endothelin-1 (ET-1) were examined in adult heart cells. The activity of mitogen-activated protein kinases (MAPKs) was measured in cytosolic extracts of isolated adult feline cardiac myocytes incubated with and without ET-1. Kinase activity was assessed by phosphorylation of the exogenous substrate, myelin basic protein. ET-1 stimulated the activity of MAPK up to 4-fold, with peak activation occurring between five and ten minutes after addition of ET-1. Polyclonal antisera raised against a 14-amino acid sequence of the erk-2 gene product, a MAPK isoform, identified two major bands in cytosolic extracts of the cardiac myocytes. Partial purification of kinase activities using Mono Q anion-exchange chromatography demonstrated two major peaks of myelin basic protein kinase activity. Subsequent immunoblots of the eluted fractions demonstrated that the immunoreactive bands observed in the cytosolic extracts eluted in those fractions possessing kinase activity. Overnight pretreatment of the cardiac myocytes with 100 ng/ml pertussis toxin inhibited the ET-1 stimulated increase in MAPK activity by 50 - 70%, but did not alter stimulation by 100 nM phorbol 12-myristate 13-acetate (PMA). These data suggest that stimulation of MAPK by ET-1 may be mediated by more than one pathway. MAPK has been shown to be activated in the intracellular transmission of growth factor signals. Indicative of a growth effect in this adult heart cell model, myocytes exposed to increasing concentrations of ET-1 demonstrated a dose dependent increase in [3H]-phenylalanine incorporation into cellular protein. This response was blocked by staurosporine and partially inhibited by pretreatment with pertussis toxin, again suggesting the possible involvement of multiple early signals. These data from isolated adult cardiac myocytes further support the hypothesis that ET-1 has a role in the regulation of cardiac growth.

Electromyography with acrylic plug surface electrodes after delivery.

Pelvic floor neuromuscular function was evaluated with surface electromyography using acrylic plug electrodes and interactions between neuromuscular function and factors pertinent to the delivery process were explored. Fifty-two women who were primiparas participated in this prospective cohort study. Circumvaginal and circumrectal muscles were assessed. Corrected vaginal-rectal flicks and holds were calculated. The results were compared by delivery route, birthweight, race, lactational status, and delivery anesthetic and to a group of nulliparous subjects. The mean interval from delivery was 46.3 days. Birthweight, race, lactational status, and anesthetic technique were not associated with statistically significant changes in electrical activity, although our ability to detect such differences was low due to the small number of subjects. Women who delivered vaginally had lower vaginal flick voltage than those delivering abdominally. Women who delivered vaginally had lower vaginal flick and hold voltages and rectal flick voltage when compared with nulliparous women studied earlier. Abdominally delivered women had values similar to the nulliparous group. Women delivering vaginally had less surface electromyographic activity in the circumvaginal muscles, implying that vaginal delivery impairs the neuromuscular function of the pelvic floor.

Activations of mitogen-activated protein kinases and phospholipase D in A7r5 vascular smooth muscle cells.

Activation of mitogen-activated protein kinases (MAPKs) was examined in the A7r5 rat vascular smooth muscle cell line. Treatment of A7r5 cells with vasopressin, phorbol ester (PMA), or serum resulted in activation of two MAPKs, Erk-1 and Erk-2. Phosphatidylinositol-specific phospholipase C was activated in response to vasopressin but not to PMA. Vasopressin and PMA both caused maximal activation of PLD within 5 minutes. Application of bacterial phospholipase D (PLD) to A7r5 cells increased phosphatidic acid to levels similar to those seen with vasopressin or PMA. Acute exposure of the cells to vasopressin, PMA, or PLD increased phosphorylation of many of the same cytosolic and membrane proteins. However, bacterial PLD did not promote significant activation of Erk-1 and Erk-2. Phosphatidic acid and lysophosphatidic acid (LPA) likewise did not stimulate MAPK activity in A7r5 cells. Serum and vasopressin stimulated DNA synthesis when present for more than 30 min, while PLD, PMA, phosphatidic acid, and LPA were not mitogenic. These data suggest that activations of MAPKs and PLD are concurrent but independent responses to vasopressin in A7r5 cells. Acute activation of these enzymes is not sufficient to simulate DNA synthesis.

Endothelin stimulates multiple responses in isolated adult ventricular cardiac myocytes.

We have examined the responses to endothelin (ET) in isolated adult cardiac myocytes from both rodent and feline species to assess whether endothelin may have a role in the induction or mediation of cardiac hypertrophy in the adult animal. We have evidence that ET acts by more than one mechanism to promote cell-signaling events believed important in growth regulation. In isolated adult cardiac ventricular myocytes labeled overnight with [3H]inositol, endothelin (ET) promoted a two- to fourfold increase in the accumulation of inositol polyphosphates in a dose-dependent manner with an half-maximal effective concentration (EC50) of approximately 5 nM. In contrast, picomolar concentrations of ET promoted an increase in both the extent and velocity of sarcomere shortening in electrically stimulated myocytes. Pretreatment of cells with pertussis toxin had no effect on the ET-stimulated phosphoinositide hydrolysis, but blocked the ET-stimulated positive inotropic effect. In addition to these early responses to ET, results obtained by Northern blot analysis demonstrate that exposure of isolated cardiac myocytes to 100 nM ET promoted the expression of c-fos and c-zif in both mammalian species. These data demonstrate that ET stimulates multiple cell-signaling pathways in adult mammalian cardiac myocytes. A paracrine mechanism of regulation of adult myocardium is suggested.

Thrombocytosis in Henoch-Schonlein syndrome.

To ascertain the frequency of thrombocytosis in Henoch-Schonlein syndrome (HSS) a small retrospective survey was undertaken. Sixty-five per cent of the cases of HSS showed thrombocytosis, which might be related to the severity of the illness.

Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization.

Thrombin stimulates polyphosphoinositide hydrolysis in embryonic chick heart cells and in 1321N1 astrocytoma cells and increases intracellular Ca2+ in the 1321N1 cells. The serine protease trypsin mimics these actions in a dose-dependent fashion, whereas the proteolytically inactive thrombin derivatives diisopropyl fluorophosphate-thrombin (DIP-thrombin) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin (PPACK-thrombin) are ineffective in this regard. The phosphoinositide responses to thrombin or trypsin and the muscarinic agonist carbachol are additive, but no additivity is observed between the responses to thrombin and trypsin. Unlike the response to carbachol, the phosphoinositide and Ca2+ responses to thrombin and trypsin desensitize, with no recovery of the calcium response even when Ca2+ stores are replenished. Cross-desensitization of phospholipase C activation and calcium mobilization between these proteases is also observed. In addition, PPACK-thrombin, which elicits no response itself, effectively inhibits trypsin-stimulated phosphoinositide hydrolysis. It is proposed that thrombin and trypsin act through the same receptor. Proteolysis appears to be important in the mechanism by which these agonists elicit phosphoinositide hydrolysis, calcium mobilization, and, perhaps, subsequent receptor desensitization.

Guanine nucleotide-dependent inositol trisphosphate formation in chick heart cells.

Stimulation of muscarinic receptors in dissociated embryonic chick heart cells promotes the hydrolysis of the phosphoinositides resulting in accumulation of the breakdown products inositol trisphosphate, bisphosphate, and monophosphate (InsP3, Insp2, and InsP, respectively). [3H]InsP3 and [3H]InsP2 are significantly elevated within 10 seconds of carbachol addition, while there is a lag in the accumulation of [3H]InsP. The time courses of the formation of the inositol phosphates suggest that carbachol activates a polyphosphoinositide-specific phospholipase C resulting in the formation of InsP3, which is subsequently metabolized to InsP2 and InsP. High-performance liquid chromotography analysis demonstrates the formation of both naturally occurring InsP3 isomers (Ins-1,4,5-P3 and Ins-1,3,4,-P3) and of inositol tetrakisphosphate (InsP4) as well. To investigate whether a guanine nucleotide-binding protein couples receptor stimulation to phosphoinositide (PI) hydrolysis in the heart, we developed a saponin-permeabilized cell preparation that would allow external manipulation of the intracellular guanosine triphosphate (GTP) concentration. In the permeabilized cell preparation, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) stimulates the accumulation of [3H]InsP, [3H]InsP2, [3H]InsP3, and [3H]InsP4. The effect of GTP gamma S is half-maximal at 1 microM and maximal above 100 microM. In contrast, GTP gamma S is ineffective in promoting PI hydrolysis in the nonpermeabilized cell except at high concentrations. Other guanine nucleotides also lead to the accumulation of [3H]InsP in the permeabilized cell, while 5'-adenylylimidodiphosphate does not. Carbachol also stimulates PI hydrolysis in the permeabilized cell preparation although it is less effective than in the intact cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic receptor properties of a pulmonary alveolar type II cell preparation from the adult rat.

The pulmonary alveolar type II cell synthesizes and secretes phosphatidylcholine (PC), a major component of surfactant, above basal level in response to beta-adrenergic stimulation. The investigation of the specific receptor which mediates these events was the topic of this study. Freshly isolated type II cells from adult rats were disrupted in a French pressure cell, and crude particulate fractions were recovered and used in assays for binding of the radioligand (-)-3-[125I]-iodocyanopindolol. The receptor had high affinity for beta-adrenergic agents, and specific binding to the receptor was saturable and reversible. The KD value obtained by kinetic means (19.6 pM) was in close agreement with that obtained by Scatchard (21.5 pM) and Hill (21.3 pM) analyses of steady-state binding data. The Scatchard correlation coefficients and Hill plot coefficients were close to 1, indicative of a single class of binding sites which displays no cooperativity. The specificity for catecholamine agonists and stereoselectivity observed were appropriate for a beta-adrenergic receptor. Use of selective drugs identified the presence of both beta 1- and beta 2-adrenergic receptor subtypes (1:3, respectively) on this cell type.

Further observations on ultrastructural changes in hepatocytes of mice infected with Schistosoma mansoni.

The effect of hepatic granulomas initiated by eggs of Schistosoma mansoni on the ultrastructure of hepatocytes of murine hosts was studied. Specimens of infected livers were collected at half week intervals, starting at week 7 postinfection and terminating at week 9 postinfection. Only the hepatocytes adjacent to granulomas showed any alteration in structure. The most obvious change was the proliferation of smooth-surfaced endoplasmic reticulum, especially striking in samples collected 8 1/2 and 9 weeks postinfection. The hypothesis was presented that the material secreted by the egg of the organism might be responsible for what appeared to be a morphologic detoxification response by the hepatocytes. Other alterations evident in the hepatocytes were an increase in the lysosomal population, mitochondrial changes and a slight hypertrophy of the Golgi complexes. Previous related studies by other investigators were explored and the findings compared with the results of this study.

Sodium valproate in the prophylaxis of simple febrile convulsions.

Thirty children with simple febrile convulsions were treated with sodium valproate following their second convulsion. Twenty-two of the 30 (73%) had no further convulsions during the one-year period of observation compared with 17 of 28 in the control group (61%). This was not a statistically significant difference. Side effects attributed to sodium valproate treatment were noted in 7 patients (23%), although 4 of these showed only mild transient gastrointestinal symptoms at high dosage. The study did not confirm any advantage in the use of sodium valproate as a prophylaxis for febrile convulsions, although compliance was good and significant side effects infrequent.