Interactions between nitric oxide and renal nerves on pressure-diuresis and natriuresis

The present study examined the effect of renal denervation on the impairment of the pressure-diuresis response produced by nitric oxide synthesis blockade. The experiments were performed in Inactin-anesthetized Munich-Wistar rats. The animals with innervated kidneys had lower baseline values of renal blood flow, GFR, sodium excretion (UNaV), and urine flow (V) than rats with denervated kidneys. Also, renal denervation shifted pressure-diuresis and natriuresis toward lower pressures. A low dose of N(omega)-nitro-L-arginine methyl esther (NAME, 3.7 nmol/kg per min) reduced UNaV and the fractional excretion of sodium (FENa) and blunted pressure-natriuresis only in rats with innervated kidneys, whereas it had no effects in rats with denervated kidneys. A medium dose of NAME (37 nmol/kg per min) lowered FENa only in rats with innervated kidneys. The administration of NAME (37 nmol/kg per min) blunted pressure-diuresis and natriuresis in kidneys with or without the renal nerves, but the effect was more pronounced in rats with innervated kidneys. A high dose of NAME (3.7 micromol + 185 nmol/kg per min) increased UNaV and FENa only in rats with innervated kidneys, whereas it reduced GFR, V, UnaV, and FENa in rats with denervated kidneys. However, pressure-natriuresis and diuresis were blunted by this high dose of NAME independently of the presence or absence of renal nerves. These results demonstrate that renal nerves potentiate the renal effects of low doses of NAME on renal function and pressure-diuresis and natriuresis. However, high doses of NAME abolish pressure-diuresis independently of renal nerves, and the natriuretic effect of NAME in innervated kidneys may be attributed to reflex inhibition of sympathetic tone due to the rise in arterial pressure.     

Prolactin plasma levels and D2-dopamine receptor occupancy measured with IBZM-SPECT

A case of primary cardiac chondrosarcoma in a 41-year-old woman who presented with cardiac tamponade and cardiac intracavitary obstruction is described. The tumor originated from the right atrium and invaded the adjacent right ventricular wall and interatrial septum. Primary cardiac chondrosarcoma is extremely rare, and its clinical, computed tomographic, echocardiographic, and magnetic resonance imaging findings are described.     

D1 and D2 dopamine receptor function in the striatum: coactivation of D1- and D2-dopamine receptors on separate populations of neurons results in potentiated immediate early gene response in D1-containing neurons

D1- and D2-dopamine receptor-mediated regulation of immediate early gene levels in identified populations of neurons in the striatum was examined with quantitative in situ hybridization histochemical techniques. Levels of messenger RNA (mRNA) encoding the immediate early genes zif268 and c-fos were examined in two experiments in rats with unilateral lesions of the nigrostriatal dopamine pathway. In a dose-response study, animals were treated with doses of 0.5, 1.0, and 1.5 mg/kg of the D1 agonist SKF-38393 either alone or in combination with the D2 agonist quinpirole (1 mg/kg). Levels of immediate early gene mRNAs 60 min following drug treatments showed a dose-related increase to the D1 agonist alone and a potentiation to combined D1 and D2 against treatment. In a second experiment, in animals receiving 1 mg/kg SKF-38393 either alone or in combination with 1 mg/kg quinpirole, the level of zif268 mRNA was measured with a double-labeling method in striatal neurons containing enkephalin mRNA, a marker of D2-containing neurons, and in neurons not containing enkephalin, putative D1-containing neurons. In the dopamine-depleted striatum, D1 agonist treatment alone did not affect enkephalin-positive neurons but significantly elevated zif268 mRNA levels in nearly all enkephalin-negative neurons. Combined D1 and D2 agonist treatment further increased zif268 mRNA levels in this population of enkephalin-negative neurons and decreased zif-268 mRNA levels in enkephalin-positive neurons. These data indicate that the synergistic response to combined D1- and D2-receptor stimulation is mediated by interneuronal interactions involving the activation of D1 and D2 receptors on separate populations of striatal neurons.     

Adenosine A1 receptor-mediated modulation of dopamine D1 receptors in stably cotransfected fibroblast cells

The antagonistic interactions between adenosine A1 and dopamine D1 receptors were studied in a mouse Ltk- cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In membrane preparations, both the adenosine A1 receptor agonist N6-cyclopentyladenosine and the GTP analogue guanyl-5'-yl imidodiphospate induced a decrease in the proportion of dopamine D1 receptors in a high affinity state. In the cotransfected cells, the adenosine A1 agonist induced a concentration-dependent inhibition of dopamine-induced cAMP accumulation. Blockade of adenosine A1 receptor signal transduction with the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or with pertussis toxin pretreatment increased both basal and dopamine-stimulated cAMP levels, indicating the existence of tonic adenosine A1 receptor activation. Pretreatment with pertussis toxin also counteracted the effects of low concentrations of the A1 agonist on D1 receptor-agonist binding. The results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the generation of second messengers.     

Interaction of D2-dopamine receptor with two pertussis toxin sensitive G proteins in human placenta

We have demonstrated the presence in human placenta of D2 dopamine receptors (D2R) which inhibit human placental lactogen (hPL) release. This inhibitory effect of dopamine (DA) was sensitive to pertussis toxin (PTX) indicating that it may be mediated by the Gi/Go family of G proteins. However, nothing is known on this G proteins/D2R interaction in human placenta. In this study, we demonstrate that DA (10(-4) M) inhibits by 39% the ADP-ribosylation by PTX of two G proteins of 40 and 41 kDa. This inhibition is receptor specific since it is reversed by spiperone, a D2R antagonist. Moreover we show that bromocriptine, a D2 agonist, inhibited the labeling of these two proteins in a dose-dependent manner with a maximal inhibition of 37% at a concentration of 10(-6) M. In order to understand the role of D2R in placental endocrinology, we have analyzed the interactions of these two PTX-sensitive G proteins with D2R in normal and abnormal pregnancies. The autoradiographs of both PTX ADP-ribosylated placental proteins of 40 and 41 kDa showed differential labeling during normal pregnancy. Thus, the relative levels of ADP-ribosylation by PTX of both proteins were 2.5 and 3.0 fold lower at term than those observed during first and second trimester whereas no difference was observed between the first and second trimester. Also, no significant change in the level of inhibition by DA was observed between 7-9 weeks and 18-40 weeks of pregnancies (35-45% inhibition). However, we observed a maximal inhibition between 10 to 17 weeks of pregnancy (64% inhibition). In placentas from preeclamptic pregnancies, the levels of ADP-ribosylation were similar to those observed in normal pregnancy, while the DA inhibition was increased by 24%. The levels of ADP-ribosylation in molar placentas reached 20% of normal values, while no difference in DA inhibition was observed. This study demonstrates that two distinct PTX-sensitive G proteins are coupled to human placental D2R. The physiological significance of the variations in these ADP-ribosylated-G proteins/D2R interaction during normal and preeclamptic pregnancies remains to be investigated.     

Fc receptor-mediated phagocytosis requires CDC42 and Rac1

At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.     

PET measurement of dopamine D2 receptor-mediated changes in striatopallidal function

This study was designed to validate an in vivo measurement of the functional sensitivity of basal ganglia neuronal circuits containing dopamine D2 receptors. We hypothesized that a D2 agonist would decrease striatopallidal neuronal activity, and hence regional cerebral blood flow (rCBF) over the axon terminals in the globus pallidus. Quantitative pallidal blood flow was measured using positron emission tomography (PET) with bolus injections of H215O and arterial sampling in six baboons before and after intravenous administration of the selective D2 agonist U91356a. We also tested whether the response to U91356a was modified by previous acute administration of various antagonists. Another baboon had serial measurements of blood flow under identical conditions, but received no dopaminergic drugs. In all animals that received U91356a, pallidal flow decreased in a dose-related manner. Global CBF had a similar response, but the decline in pallidal flow was greater in magnitude and remained significant after accounting for the global effect. A D2 antagonist, but not antagonists of D1, serotonin-2, or peripheral D2 receptors, prevented this decrease. This work demonstrates and validates an in vivo measure of the sensitivity of D2-mediated basal ganglia pathways. It also supports the hypothesis that activation of the indirect striatopallidal pathway, previously demonstrated using nonselective D2-like agonists, can be mediated specifically by D2 receptors. We speculate that the U91356a-PET technique may prove useful in detecting functional abnormalities of D2-mediated dopaminergic function in diseases such as parkinsonism, dystonia, Tourette syndrome, or schizophrenia.     

Pressure diuresis and natriuresis in DOCA-salt mice

The increasing use of mice in renal and cardiovascular studies has necessitated adapting physiological methods used for rats to mice, which are far smaller in size. We have adapted measurements of continuous renal blood flow, pressure natriuresis and diuresis, and laser-Doppler cortical and medullary flow to 40 g mice with DOCA-salt hypertension. We demonstrated a rightward shift in the pressure-natriuresis-diuresis curve. We conclude that with current, commercially-available equipment, sophisticated renal physiology can be conducted in the mouse. These methods will be important to investigations of gene-targeted mice.     

Variability in D2-dopamine receptor density and affinity: a PET study with [11C]raclopride in man

The variability of D2-dopamine receptor binding parameters in man was determined using Positron Emission Tomography (PET) and [11C]raclopride. A saturation analysis based on five PET-experiments was performed in each of ten men and ten women. The mean density of D2-dopamine receptors (Bmax) was 28 +/- 6.9 pmol/ml (mean +/- S.D.) and the apparent affinity (Kdapp) 9.1 +/- 1.9 pmol/ml. The Hill coefficient was in all subjects close to unity (nH: 0.999 +/- 0.020), thereby indicating binding to a homogeneous class of receptors. No significant differences between males and females were found in Bmax or Kdapp. The interindividual difference in Bmax was statistically significant (alpha = 0.01). The difference in Kdapp was not significant. Upregulation of the receptor density (Bmax) has been widely discussed as a mechanism for increased dopaminergic neurotransmission in schizophrenia. This study indicates that receptor density varies considerably in a group of healthy subjects.     

Renal nerves are not involved in sodium and water retention during mechanical ventilation in awake dogs

BACKGROUND: The role of renal nerves during positive end-expiratory pressure ventilation (PEEP) has only been investigated in surgically stressed, anesthetized, unilaterally denervated dogs. Anesthesia, sedation, and surgical stress, however, decrease urine volume and sodium excretion and increase renal sympathetic nerve activity independent of PEEP. This study investigated in awake dogs the participation of renal nerves in mediating volume and water retention during PEEP. METHODS: Eight tracheotomized, trained, awake dogs were used. The protocol consisted of 60 min of spontaneous breathing at a continuous positive airway pressure of 4 cm H2O, followed by 120 min of controlled mechanical ventilation with a mean PEEP of 15-17 cm H2O (PEEP), and 60 min of continuous positive airway pressure. Two protocols were performed on intact dogs, in which volume expansion had (hypervolemic; electrolyte solution, 0.5 ml x kg(-1) x min(-1)) and had not (normovolemic) been instituted. This was repeated on the same dogs 2 or 3 weeks after bilateral renal denervation. RESULTS: Hypervolemic dogs excreted more sodium and water than did normovolemic dogs. There was no difference between intact and renal-denervated dogs. Arterial pressure did not decrease when continuous positive airway pressure was switched to PEEP. Plasma renin activity, aldosterone, and antidiuretic hormone concentrations were greater in normovolemic dogs. The PEEP increased aldosterone and antidiuretic hormone concentrations only in normovolemic dogs. CONCLUSIONS: In conscious dogs, renal nerves have no appreciable contribution to sodium and water retention during PEEP. Retention in normovolemic dogs seems to be primarily caused by an activation of the renin-angiotensin system and an increase in the antidiuretic hormone. Excretion rates depended on the volume status of the dogs.     

Dopamine D2 receptor-mediated modulation of rod-cone coupling in the Xenopus retina

Lectins are able to bind to cholecystokinin (CCK) receptors and other glycosylated membrane proteins. The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) are used for affinity chromatography to isolate the highly glycosylated CCK-A receptor of pancreatic acinar cells. According to the working hypothesis that lectin binding to the CCK receptor should alter the ligand-receptor interaction, the effect of WGA and UEA-I on CCK-8-induced enzyme secretion was studied on isolated rat pancreatic acini in vitro. In vitro both lectins showed a dosage-dependent inhibition of CCK-8-induced alpha-amylase secretion of acini over 60 min. WGA showed a strong inhibitory effect on amylase secretion, approximately 40%, in vitro. UEA-I caused a smaller, but significant decrease, approximately 20%, in enzyme secretion of isolated acini. Additionally, both lectins inhibited cerulein/secretin- or cerulein-induced pancreatic secretion of rats in vivo, but not after secretin alone. The results are discussed with respect to a possible influence of both lectins on the interaction of CCK or cerulein with the CCK-A receptor.     

D4 dopamine receptor-mediated signaling events determined in transfected Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line stably expressing a recombinant human D4 dopamine receptor made from a synthetic gene has been used to determine potential D4-mediated signaling events. We designed and synthesized a modified gene coding for a human D4 receptor with reduced G + C content but unaltered encoded amino acids. Stable expression of this gene was obtained in two cell lines, inducible expression in CHO lacI cells and constitutive expression in HEK293 cells. In CHO lacI cells induced to express D4 receptors but not in uninduced cells, dopamine and quinpirole inhibit forskolin-stimulated cAMP accumulation and potentiate ATP-stimulated [3H]arachidonic acid release through a mechanism that requires protein kinase C but is unaffected by membrane-soluble cAMP analogs. In addition, D4 receptor activation causes an increase in the rate of extracellular acidification measured by microphysiometry. This response is unaffected by protein kinase C down-regulation but is inhibited by removal of extracellular sodium and inhibitors of NaH-1 exchange, suggesting the involvement of a Na+/H+ exchanger. All responses are blocked by clozapine and are sensitive to pertussis toxin. D4 receptors, like other G(i)/G(o)-linked receptors, mediate multiple signaling events, and the pathways activated are similar to those used by D2 and D3 receptors expressed in similar cells.     

Ethanol inhibition of AMPA and kainate receptor-mediated depolarizations of hippocampal area CA1

Longitudinal hippocampal slices were prepared from adult female rats. The excitatory amino acids, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainic acid, were applied to area CA1, and the resulting depolarizations were measured using the grease-gap electrophysiological technique. Agonist dose-response curves were generated in the presence and absence of various concentrations of ethanol. Ethanol (25-200 mM) significantly attenuated the depolarizations that were produced by each agonist. In addition, we found that ethanol potently antagonized kainate-induced depolarizations across the agonist concentration-response curve, whereas it significantly suppressed only AMPA responses that were induced with moderate-to-high agonist concentrations. These results indicate that ethanol has potent antagonist actions against non-N-methyl-D-aspartate (NMDA) excitatory amino acid-induced neuronal depolarizations in hippocampal area CA1. Moreover, the relative potency of ethanol depends on the specific excitatory agonist tested and the concentration of that agonist. This suggests that, in addition to the known effects of ethanol on NMDA receptor-mediated activity, it may also potently attenuate ongoing "fast" glutamatergic synaptic activity in the hippocampus.     

Intrarenal nitric oxide activity and pressure natriuresis in anesthetized dogs

In the course of an ongoing cohort study on constitutional and occupational risk factors for the development of irritant hand dermatitis in hairdressing apprentices, an increased prevalence of irritant skin changes was noted in a subgroup examined during particularly cold winter months. Prompted by this observation, the importance of several meteorological factors (day means of temperature, relative and absolute humidity) was assessed in extensive statistical analyses based on data of 742 participants, supplemented by meteorological information obtained from the German Meteorological Service (DWD). There were significant associations of existing hand dermatitis with low temperature and low absolute humidity (Mann-Whitney U-test, P < 0.0001), but not with relative humidity (P = 0.38). Logistic regression analysis, including known determinants of irritant hand dermatitis in this setting, showed that low temperature and low relative humidity tended to be risk factors (OR = 1.66 and 1.57, respectively, for the lower quartiles, P = 0.07 in both cases), and confirmed that absolute humidity significantly influenced the occurrence of irritant hand dermatitis (OR = 2.06 for < 4.8 mg/L, P < 0.01). Thus, these environmental factors must be regarded as possible confounders in the analysis of future epidemiological studies on irritant hand dermatitis and should be considered in multifactorial analyses.     

Enhanced kappa-opioid receptor-mediated analgesia by antisense targeting the sigma1 receptor

In the current study, we used an antisense oligodeoxynucleotide targeting the recently cloned sigma1 receptor to assess its functions within the nervous system. Sigma1 antagonists potentiate the analgesic actions of opioids. Similarly, the antisense probe targeting the sigma1 receptor enhanced the analgesic activity of the kappa1-opioid receptor agonist U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +) and the kappa3-opioid receptor agonist naloxone benzoylhydrazone. A mismatch control was inactive. These results confirm the role of sigma1 receptors in an anti-opioid analgesic system and illustrate the utility of antisense approaches towards the elucidation of sigma receptor functions.     

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.     

Augmentation of antigen receptor-mediated responses by histamine H1 receptor signaling

Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein-coupled receptors. Here, we report that histamine may affect antigen receptor-mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative responses of anti-CD3epsilon- or anti-IgM-stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit-positive cells were depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T cells had low proliferative responses to anti-CD3epsilon cross-linking or antigen stimulation in vitro. B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor-mediated immune responses, suggesting cross-talk between G protein-coupled receptors and antigen receptor-mediated signaling.     

Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch

The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification. Here we investigate the possible functional synergy of the two genes. The generation of Hoxa1(3'RARE)/Hoxb1(3'RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3'RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1(null)/Hoxb1(3'RARE) and Hoxa1(null)/Hoxb1(null )double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involuted by 10.5 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants.     

Renal and hepatic 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in piglets suffering from pseudo vitamin D-deficiency rickets, type I

Research has shown that speech articulated in a clear manner is easier to understand than conversationally spoken speech in both the auditory-only (A-only) and auditory-visual (AV) domains. Because this research has been conducted using younger adults, it is unknown whether age-related changes in auditory and/or visual processing affect older adults' ability to benefit when a talker speaks clearly. The present study examined how speaking mode (clear vs conversational) and presentation mode (A-only vs AV) influenced nonsense sentence recognition by older listeners. Results showed that neither age nor hearing loss limited the amount of benefit that older adults obtained from a talker speaking clearly. However, age was inversely correlated with identification of AV (but not A-only) conversational speech, even when pure-tone thresholds were controlled statistically.     

Alterations in alpha 1-adrenergic receptor-mediated phosphatidylinositol turnover in hypoxic cardiac myocytes

The purpose of these studies was to examine the effects of hypoxia on alpha 1-adrenergic receptor (alpha 1AR) mediated phosphatidylinositol (PI) turnover in cultured neonatal rat cardiac myocytes. Cells were pre-labeled with [3H]-inositol and incubated for 1 h in either normoxia or hypoxia. Phenylephrine, an alpha 1AR agonist, was added at various time intervals (0-60 min) before termination of the incubation. There was a time-dependent release of radioactivity from the lipid fraction to the aqueous fraction with alpha 1AR stimulation. alpha 1AR-mediated PI turnover was biphasic in normoxic cells and monophasic in hypoxic cells. Using ion-exchange chromatography, radioactivity in the inositol trisphosphate (IP3) peak was increased with acute phenylephrine stimulation (5 min) in the normoxic cells, while inositol phosphate (IP) and inositol bisphosphate (IP2) were increased with chronic stimulation (60 min). After 5 min of alpha 1AR stimulation, hypoxia did not alter total aqueous radioactivity when compared to normoxia, but there was a significant increase in IP2. However, there was decreased PI turnover in chronically stimulated (30-60 min) hypoxic cells when compared to normoxic cells. Hypoxia had no effect on radioactivity in the IP3 fraction with either 0, 5, or 60 min of alpha 1AR stimulation, but there was a significant increase in [1,4,5]-IP3 in hypoxic cells with 30 s alpha 1AR stimulation. With hypoxia, there was no difference in radioactivity in the phosphatidylinositols with either 0 or 5 min stimulation when compared to normoxia. However, after 60 min of alpha 1AR stimulation, hypoxia resulted in increased PI and PIP, when compared to normoxic cells, but PIP2 radioactivity was unchanged. There was no effect of pertussis toxin on either the acute or chronic phase of PI turnover, negating involvement of Gi or G(o). These data suggest that alpha 1AR stimulation in neonatal rat cardiac myocytes is biphasic, and that hypoxia produces a slower monophasic response during extended alpha 1-agonist exposure as would be found with ischemia.