CYP1A2 activity as measured by a caffeine test predicts clozapine and active metabolite steady-state concentrationin patients with schizophrenia.

Clozapine is an atypical antipsychotic drug and displays efficacy in 30% to 60% of patients with schizophrenia who do not respond to traditional antipsychotics. A clozapine concentration greater than 1,150 nmol/L increases the probability of antipsychotic efficacy. However, plasma clozapine concentration can vary more than 45-fold during long-term treatment. The aim of this study was to assess the contribution of CYP1A2 to variability in steady-state concentration of clozapine and its active metabolite norclozapine. Patients with schizophrenia or schizoaffective disorder were prospectively monitored during clozapine treatment (N = 18). The in vivo CYP1A2 activity was measured using the caffeine metabolic ratio (CMR) in overnight urine. Trough plasma samples were drawn after at least 5 days of treatment with a constant regimen of clozapine. A significant negative association was found between the CMR and the dose-corrected clozapine (r(s) = -0.87,p < 0.01) and norclozapine (r(s) = -0.76,p < 0.01) concentrations. Nonsmokers displayed a higher clozapine (3.2-fold) and norclozapine (2.3-fold) concentration than smokers (p < 0.05). Furthermore, there was marked person-to-person variation in CYP1A2 activity during multiple-dose clozapine treatment (coefficient of variation = 60%). Age, weight, serum creatinine, and grapefruit juice consumption did not significantly contribute to variability in clozapine and norclozapine concentration (p > 0.05). In conclusion, CYP1A2 is one of the important contributors to disposition of clozapine during multiple-dose treatment. Although further in vitro experiments are necessary, the precise metabolic pathways catalyzed by CYP1A2 seem to be subsequent to the formation of norclozapine, hitherto less recognized quantitatively important alternate disposition routes, or both. From a clinical perspective, an environmentally induced or constitutively high CYP1A2 expression can lead to a decrease in steady-state concentration of clozapine as well as its active metabolite norclozapine. Thus, interindividual variability in CYP1A2 activity may potentially explain treatment resistance to clozapine in some patients. CYP1A2 phenotyping with a simple caffeine test may contribute to individualization of clozapine dosage and differentiate between treat ment noncompliance and high CYP1A2 activity.

Chronotropic effect of angiotensin II via type 2 receptors in rat brain neurons.

Previously, we determined that angiotensin II (Ang II) elicits an Ang II type 2 (AT(2)) receptor-mediated increase of neuronal delayed rectifier K(+) (I(KV)) current in neuronal cultures from newborn rat hypothalamus and brain stem. This requires generation of lipoxygenase (LO) metabolites of arachidonic acid (AA) and activation of serine/threonine phosphatase type 2A (PP-2A). Enhancement of I(KV) results in a decrease in net inward current during the action potential (AP) upstroke as well as shortening of the refractory period, which may lead to alterations in neuronal firing rate. Thus, in the present study, we used whole-cell current clamp recording methods to investigate the AT(2) receptor-mediated effects of Ang II on the firing rate of cultured neurons from the hypothalamus and brain stem. At room temperature, these neurons exhibited spontaneous APs with an amplitude of 77.72 +/- 2.7 mV (n = 20) and they fired at a frequency of 0.8 +/- 0.1 Hz (n = 11). Most cells had a prolonged early after-depolarization that followed an initial fully developed AP. Superfusion of Ang II (100 nM) plus losartan (LOS, 1 microM) to block Ang II type 1 receptors elicited a significant chronotropic effect that was reversed by the AT(2) receptor inhibitor PD 123,319 (1 microM). LOS alone had no effect on any of the parameters measured. The chronotropic effect of Ang II was reversed by the general LO inhibitor 5,8,11,14-eicosatetraynoic acid (10 microM) or by the selective PP-2A inhibitor okadaic acid (1 nM) and was mimicked by the 12-LO metabolite of AA 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraynoic acid. These data indicate that Ang II elicits an AT(2) receptor-mediated increase in neuronal firing rate, an effect that involves generation of LO metabolites of AA and activation of PP-2A.

Treatment-resistance to clozapine in association with ultrarapid CYP1A2 activity and the C-->A polymorphism in intron 1 of the CYP1A2 gene: effect of grapefruit juice and low-dose fluvoxamine.

Antipsychotic response to clozapine varies markedly among patients with schizophrenia. The disposition of clozapine is dependent, in part, on the cytochrome P-450 (CYP) 1A2 enzyme in vivo. In theory, a very high CYP1A2 activity may lead to subtherapeutic concentrations and treatment resistance to clozapine. This prospective case study evaluates the clinical significance of ultrarapid CYP1A2 activity and a recently discovered single nucleotide (C --> A) polymorphism in intron 1 of the CYP1A2 gene (CYP1A2*F) for treatment resistance to clozapine. In addition, we describe the effect of grapefruit juice or low-dose fluvoxamine (25-50 mg/d) coadministration on clozapine and active metabolite norclozapine steady-state plasma concentration and antipsychotic response.

Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulin-dependent protein kinase II.

Angiotensin II (Ang II) acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. These actions of Ang II are mediated via Ang II type 1 (AT1) receptors and involve modulation of membrane ionic currents and neuronal activity. In previous studies we utilized neurons cultured from the hypothalamus and brain stem of newborn rats to investigate the AT1 receptor-mediated effects of Ang II on neuronal K+ currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to activation of protein kinase C (PKC), specifically PKCalpha. However, the data also indicated that another Ca2+-dependent mechanism was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a known modulator of K+ currents in neurons, we investigated the role of this enzyme in the AT1 receptor-mediated reduction of neuronal Kv current by Ang II. The reduction of neuronal Kv current by Ang II was attenuated by selective inhibition of either calmodulin or CaM KII and was mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIalpha. Concurrent inhibition of CaM KII and PKC completely abolished the reduction of neuronal Kv by Ang II. Consistent with these findings is the demonstration that Ang II increases CaM KII activity in neuronal cultures, as evidenced by increased levels of autophosphorylated CaM KIIalpha subunit. Last, single-cell reverse transcriptase (RT)-PCR analysis revealed the presence of AT1 receptor-, CaM KIIalpha-, and PKCalpha subunit mRNAs in neurons that responded to Ang II with a decrease in Kv current. The present data indicate that the AT1 receptor-mediated reduction of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII pathway, in addition to the previously documented involvement of PKC.

Angiotensin II type 2 receptor stimulation of neuronal delayed-rectifier potassium current involves phospholipase A2 and arachidonic acid.

Angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor-mediated increase in delayed-rectifier K+ current (IK) in neurons cultured from newborn rat hypothalamus and brainstem. This effect involves a pertussis toxin (PTX)-sensitive Gi protein and is abolished by inhibition of serine and threonine phosphatase 2A (PP-2A). Here, we determined that Ang II stimulates [3H]arachidonic acid (AA) release from cultured neurons via AT2 receptors. This effect of Ang II was blocked by inhibition of phospholipase A2 (PLA2) and by PTX. Because AA and its metabolites are powerful modulators of neuronal K+ currents, we investigated the involvement of PLA2 and AA in the AT2 receptor-mediated stimulation of IK by Ang II. Single-cell reverse transcriptase (RT)-PCR analyses revealed the presence of PLA2 mRNA in neurons that responded to Ang II with an increase in IK. The stimulation of neuronal IK by Ang II was attenuated by selective inhibitors of PLA2 and was mimicked by application of AA to neurons. Inhibition of lipoxygenase (LO) enzymes significantly reduced both Ang II- and AA-stimulated IK, and the 12-LO metabolite of AA 12S-hydroxyeicosatetraenoic acid (12S-HETE) stimulated IK. These data indicate the involvement of a PLA2, AA, and LO metabolite intracellular pathway in the AT2 receptor-mediated stimulation of neuronal IK by Ang II. Furthermore, the demonstration that inhibition of PP-2A abolished the stimulatory effects of Ang II, AA, and 12S-HETE on neuronal IK but did not alter Ang II-stimulated [3H]-AA release suggests that PP-2A is a distal event in this pathway.

Modulation of K+ and Ca2+ currents in cultured neurons by an angiotensin II type 1a receptor peptide.

Angiotensin II (ANG II) inhibits delayed rectifier K+ current (IK) and stimulates total Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem, effects mediated via ANG II type 1 (AT1) receptors. Here, we identify potential G protein activator regions of the AT1 receptor responsible for initiating the intracellular changes that lead to alterations in these currents. Intracellular application into cultured neurons of a peptide corresponding to the third cytoplasmic loop of the AT1 receptor (AT1a/i3) mimicked the actions of ANG II on IK and ICa, whereas application of a peptide corresponding to the second cytoplasmic loop (AT1a/i2) did not alter these currents. This modulation of IK and ICa by AT1a/i3 involves intracellular messengers (G alpha q, protein kinase C, and intracellular Ca2+) that are identical to those involved in the modulation of IK and ICa following ANG II activation of AT1 receptors. These data provide functional evidence for a role of the third cytoplasmic loop of the AT1 receptor in G protein coupling and subsequent modulation of ion channel effectors.

Mechanisms underlying the chronotropic effect of angiotensin II on cultured neurons from rat hypothalamus and brain stem.

The chronotropic effect of angiotensin II (Ang II) was studied in cultured neurons from rat hypothalamus and brain stem with the use of the patch-clamp technique. Ang II (100 nM) increased the neuronal spontaneous firing rate from 0.8 +/- 0.3 (SE) Hz in control to 1.3 +/- 0.4 Hz (n = 7, P < 0.05). The amplitude of threshold stimulation was decreased by Ang II (100 nM) from 82 +/- 4 pA to 62 +/- 5 pA (n = 4, P < 0.05). These actions of Ang II were reversed by the angiotensin type 1 (AT1) receptor antagonist losartan (1 microM). In the presence of tetrodotoxin, Ang II (100 nM) significantly increased the frequency and the amplitude of the Cd2+-sensitive subthreshold activity of the cultured neurons. Ang II also stimulated the subthreshold early afterdepolarizations (EADs) to become fully developed action potentials. Similar to the action of Ang II, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) increased the firing rate from 0.76 +/- 0.3 Hz to 2.3 +/- 0.5 Hz (n = 6, P < 0.05) and increased the neuronal subthreshold activity. After neurons were intracellularly dialyzed with PKC inhibitory peptide (PKCIP, 5 microM), PMA alone, Ang II alone, or PMA plus Ang II no longer increased the action potential firing initiated from the resting membrane potential level. However, superfusion of PMA plus Ang II or Ang II alone increased the number of EADs that reached threshold and produced action potentials even in the presence of PKCIP (5 microM, n = 4). The actions of Ang II could also be mimicked by depolarizing pulse and K+ channel blockers (tetraethylammonium chloride or 4-aminopyridine). These results indicate that Ang II by activation of AT1 receptors increases neuronal excitability and firing frequency, and that this may involve both PKC dependent and -independent mechanisms.

A-type K+ current in neurons cultured from neonatal rat hypothalamus and brain stem: modulation by angiotensin II.

The regulation of A-type K+ current (I(A)) and the single channel underlying I(A) in neonatal rat hypothalamus/brain stem cultured neurons were studied with the use of the patch-clamp technique. I(A) had a threshold of activation between -30 and -25 mV (n = 14). Steady-state inactivation of I(A) occurred between -80 and -70 mV and had a membrane voltage at which I(A) was half-maximum of -52.2 mV (n = 14). The mean values for the activation and inactivation (decay) time constants during a voltage step to +20 mV were 2.1 +/- 0.3 (SE) ms (n = 8) and 13.6 +/- 1.9 ms (n = 8), respectively. Single-channel recordings from outside-out patches revealed A-type K+ channels with voltage-dependent activation, 4-aminopyridine (4-AP) sensitivity, and inactivation kinetics similar to those of I(A). The single-channel conductance obtained from cell-attached patches was 15.8 +/- 1.3 pS (n = 4) in a physiological K+ gradient and 41.2 +/- 3.7 pS (n = 5) in symmetrical 140 mM K+. Angiotensin II (Ang II, 100 nM) reduced peak I(A) by approximately 20% during a voltage step to +20 mV (n = 8). Similarly, Ang II (100 nM) markedly reduced single A-type K+ channel activity by decreasing open probability (n = 4). The actions of Ang II on I(A) and single A-type K+ channels were reversible either by addition of the selective angiotensin type 1 (AT1) receptor antagonist losartan (1 microM) or on washout of the peptide. Thus the activation of AT1 receptors inhibits a tetraethylammonium-chloride-resistant, 4-AP-sensitive I(A) and single A-type K+ channels, and this may underlie some of the actions of Ang II on electrical activity of the brain.

Functional interactions between neuronal AT1 and AT2 receptors.

Angiotensin II (Ang II), via the activation of the AT1 and AT2 receptors regulates electrophysiological responses of catecholaminergic neurons. This study was designed to determine if functional interactions between AT1 and AT2 receptors exist in a single neuron. Ang II caused two unique electrophysiological responses characteristic of receptor crosstalk. First, Ang II elicited an AT1 receptor-mediated decrease in I(K) followed by an AT2 receptor-mediated increase in I(K). Second, Ang II elicited an AT2 receptor-mediated increase in I(K) followed by an AT1 receptor-mediated decrease in I(K). AT1 and AT2 receptors were co-localized on the catecholaminergic neurons. These observations suggest, for the first time, the existence of a crosstalk between Ang II receptor subtypes that may be significant in the physiological activity of catecholaminergic neurons.

Angiotensin II regulation of intracellular calcium in astroglia cultured from rat hypothalamus and brainstem.

This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1-100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura-2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl2), the Ang II-stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady-state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 microM) inhibited the Ang II-stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 microM) did not inhibit the Ang II-stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II-stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca(2+)-free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 microM), cyclopiazonic acid (10 microM), and ryanodine (10 microM), but not caffeine (1-10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 microM) and nifedipine (10 microM); in contrast, gadolinium (100 microM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.

Angiotensin II type 2 receptor-mediated regulation of rat neuronal K+ channels.

We have previously shown that angiotensin II (Ang II), via AT2 receptors, increases whole-cell K+ current in cultured rat hypothalamus and brain stern neurons. We have now investigated the AT2 receptor-mediated effects of Ang II on the activity of single delayed rectifier K+ channels in cell-attached membrane patches. In control recordings (bath, 5.4 mmol/L K+; pipette, 140 mmol/L K+), two voltage-dependent channels were recorded with conductances of 34 +/- 4 and 56 +/- 6 pS, respectively (n = 6). When patches were excised, the channels reversed near a membrane potential expected for a K+ channel. In cell-attached patches (-40 mV), Ang II (100 nmol/L) increased open probability of the 56-pS K+ channel from 0.03 +/- 0.01 to 0.21 +/- 0.05 (n = 3). The selective AT2 receptor antagonist PD 123319 (1 mumol/L) but not the AT1 receptor antagonist losartan (1 mumol/L) blocked the actions of Ang II (n = 3). The selective AT2 receptor agonist CGP 42112 (100 nmol/L) produced similar effects to Ang II. Kinetic analysis of the Ang II effect showed that open-time histograms were best fit by two exponential functions. Ang II increased both open-time constants relative to control (control, tau 1 = 0.9 +/- 0.1 milliseconds, tau 2 = 2.3 +/- 0.3 milliseconds; Ang II, tau 1 = 3.1 +/- 0.4 milliseconds, tau 2 = 12.1 +/- 2.4 milliseconds), and PD 123319 blocked this effect (n = 3). The closed-time histogram was not affected by Ang II PD 123319, or losartan. These results suggest that activation of AT2 receptors modulates rat hypothalamus and brain stern neuronal whole-cell K+ current by increasing the open probability of a 56-pS K+ channel.

Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms.

Angiotensin II (ANG II) elicits an ANG II type 1 (AT1) receptor-mediated decrease in voltage-dependent K+ current (Ik) and an increase in voltage-dependent Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem. Modulation of these currents by ANG II involves intracellular messengers that result from an AT1 receptor-mediated stimulation of phosphoinositide hydrolysis. For example, the effects of ANG II on IK and ICa were abolished by phospholipase C antagonists. The reduction in IK produced by ANG II was attenuated by either protein kinase C (PKC) antagonists or by chelation of intracellular Ca2+. By contrast, PKC antagonism abolished the stimulatory effect of ANG II on ICa. Superfusion of the PKC activator phorbol 12-myristate 13-acetate produced effects on IK and ICa similar to those observed after ANG II. Furthermore, intracellular application of inositol 1,4,5-trisphosphate (IP3) elicited a significant reduction in IK. This suggests that the AT1 receptor-mediated changes in neuronal K+ and Ca2+ currents involve PKC (both IK and ICa) and IP3 and/or intracellular Ca2+ (IK).

Modulation of the delayed rectifier K+ current in neurons by an angiotensin II type 2 receptor fragment.

Angiotensin II (ANG II) stimulates the delayed rectifier K+ current (IK) in neurons cultured from rat hypothalamus and brain stem via AT2 receptors, and this effect involves activation of a Gi protein and protein phosphatase 2A (PP2A). However, there was no evidence that the AT2 receptor involved in this response was the same as the recently cloned AT2 receptor. In the present study, intracellular injection of a 22-amino acid peptide (PEP-22) corresponding to the putative third intracellular loop of the cloned AT2 receptor elicited an increase in IK in cultured neurons that was similar to the effect produced by ANG II. Furthermore, this effect of PEP-22 was abolished by pertussis toxin (200 ng/ml, 24 h) pretreatment and also by superfusion of the PP2A inhibitor okadaic acid (10 nM), suggesting the involvement of Gi protein and PP2A, respectively. Intracellular injection of a random peptide or normal pipette solution did not affect neuronal IK. This is direct evidence to link the cloned AT2 receptor to a defined response elicited by ANG II.

Calcium-modulated inward rectification of a calcium-activated potassium channel in neurons.

1. Inward rectification of a calcium-activated K+ channel in neurons cultured from the hypothalamus and brain stem of 1-day-old rats was studied by using patch-clamp techniques. A big conductance calcium-activated K+ channel with a slow gating rate was observed in inside-out patches. With symmetrical K+ across patches, inward conductance of this calcium-activated K+ channel was 216 +/- 14 (SE) pS (n = 4 patches), which changed little as different [Ca2+] was included in the bath solution. Outward conductance of this calcium-activated K+ channel was regulated by [Ca2+] in the bath solution and was 74 +/- 15 pS with 500 microM Ca2+. The higher level of Ca2+ on the intracellular side of the membrane caused the larger degree of rectification. Mg2+ only had a minor effect on rectification of this calcium-activated K+ channel.

Angiotensin II type 2 receptor stimulation of neuronal K+ currents involves an inhibitory GTP binding protein.

Angiotensin II (ANG II) elicits an ANG II type 2 (AT2) receptor-mediated increase in outward K+ current (IK; delayed rectifier K+ current) in neurons cocultured from rat hypothalamus and brain stem. Here we have shown that the AT2-receptor-mediated stimulation of neuronal IK by ANG II (100 nM) was abolished by pretreatment of cultures with pertussis toxin (PTX; 200 ng/ml) and by intracellular application of an antibody against the inhibitory guanine nucleotide (GTP) binding protein (anti-Gi alpha, 1:200). Antibodies against other GTP binding proteins (anti-Go alpha, 1:50 and 1:200; anti-Gq/11 alpha, 1:200) did not alter the AT2-receptor-mediated stimulation of neuronal IK by ANG II (100 nM). Furthermore, this effect of ANG II (100 nM) was inhibited by the serine/threonine phosphatase inhibitor okadaic acid (1-10 nM) and by anti-type 2A protein phosphatase (PP2A) antibodies but not by the tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). Thus we have identified key components (Gi and PP2A) of the signal transduction pathway that is responsible for the AT2-receptor-mediated stimulation of neuronal K+ currents.

Receptor-mediated effects of angiotensin II on neurons.

Aside from its well-known and numerous actions at peripheral tissues, the octapeptide angiotensin II (ANG II) elicits specific receptor-mediated effects within the central nervous system. In this review we focus on the receptor-mediated actions of ANG II on neurons. The distribution of ANG II receptors in the brain and physiological, electrophysiological, and cellular effects mediated by these receptors are discussed. This is extended to a review of the characteristics of ANG II receptor subtypes on cultured neurons and the cellular and genomic actions mediated by these receptors. Finally, we develop this information into speculative models for the cellular effects mediated by each ANG II receptor subtype in neurons.

Angiotensin II type 2 receptor-modulated changes in potassium currents in cultured neurons.

We have previously shown that angiotensin II (ANG II) stimulates an increase in net outward ionic current (Ino) in neurons cocultured from neonate rat hypothalamus and brain stem, an effect mediated by ANG II type 2 (AT2) receptors. Ino consists mainly of K+ and Ca2+ currents, and in the present study we used whole cell voltage clamp procedures to define which of these currents are modulated by AT2 receptors. We determined that ANG II (50-100 nM) stimulated both transient K+ current (IA) and delayed-rectifier K+ current (IK) in cultured neurons. The effects were mediated by AT2 receptors (blocked by 1 microM PD-123177 but not by 1 microM losartan). For both IA and IK, ANG II elicited an increase in maximal conductance. By contrast, ANG II altered neither Ca(2+)-activated K+ current nor Ca2+ current. Our data demonstrate discrete AT2 receptor-mediated effects of ANG II on IA and IK in cultured neonate neurons. Importantly, these data provide an electrophysiological basis for behavioral or physiological effects (as yet undefined) mediated by this ANG II receptor subtype in the brain.

Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT1 and AT2 receptors.

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I(no)) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. Ino is the sum of all inward and outward membrane currents (minus Na+, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I(no) in cultured neurons. AII caused a reversible and concentration (0.1 nM-10 microM)-dependent increase in I(no). This effect is inhibited by the AT2 receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT1-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT2 receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM-10 microM)-dependent decrease in I(no) that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I(no) is mediated by AT1 receptors. Additionally, some neurons displayed both AT1- and AT2 receptor-mediated effects on I(no). Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.

Rat atrial muscle responses with caffeine: dose-response, force frequency, and postrest contractions.

Caffeine has been reported to have a positive and (or) a negative inotropic effect on cardiac muscle. In this study, the force-frequency and postrest characteristics of rat atrium were studied in the presence of caffeine (1.0-10 mM) to see if the interval between beats affected the response of cardiac muscle to caffeine. When stimulation frequency was 0.5 or 2.0 Hz, there was a positive followed by a negative inotropic response with 1, 5, or 10 mM caffeine. Incomplete relaxation occurred under these circumstances, giving rise to contracture. At low frequency of stimulation (0.1 Hz) caffeine had only a negative inotropic effect, and this effect was greater with 1 mM caffeine than with 5 mM caffeine. In the absence of caffeine, when stimulation at 0.5 or 3 Hz was interrupted, a pause of 2-20 s resulted in potentiation. When caffeine was present (2.0 mM), postrest potentiation was severely attenuated, but the steady-state contraction amplitude within the range 0.5-3.0 Hz was not different. These results are consistent with the hypothesis that caffeine induces a leak of Ca2+ from the sarcoplasmic reticulum, and this Ca2+ is extruded from the cell, possibly by Na+/Ca2+ exchange. Sarcoplasmic reticular uptake of Ca2+ and the translocation to release sites appear not to be affected by caffeine within 1-5 mM concentrations.

Effects of chronic hypoxia during maturation on the negative chronotropic effect of [H+] on the rabbit sino-atrial node.

Previous studies in our laboratory have shown an effect of 12 weeks of hypoxia on the electrophysiology of the heart. This study was designed to determine the effects of hypoxia for shorter periods (1, 2, 3 and 6 weeks) on the neonatal heart as well as the chronotropic effects of [H+] on sino-atrial automaticity. Rabbits (2 days of age) were raised for 1, 2, 3 or 6 weeks in either a normal or hypoxic environment. At the end of 1, 2, 3 or 6 weeks, they were killed and studied using an isolated sino-atrial tissue preparation and standard microelectrode techniques. Measurements of hematocrit, ventricular weight and ventricular total protein were made. Right ventricular hypertrophy appeared at 2 and 3 weeks in the hypoxic hearts. There was no difference in left ventricular weight or total protein between normal and hypoxic groups over the time studied. Both the basal and maximal (isoproterenol stimulated) spontaneous rates decreased with age. The baseline and maximal rates from hypoxic rabbits were significantly less than those from normal rabbits at 3 and 6 weeks (p less than 0.05). The net negative chronotropic effect of elevated [H+] was more pronounced in the hypoxic than the normal preparations at 3 and 6 weeks. In summary, neonates raised in hypoxia for as short a period as 3 weeks show right ventricular hypertrophy and a decrease in sino-atrial automaticity. The net negative chronotropic effect of [H+] is enhanced with development and chronic hypoxia.