Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells.

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [32P]phosphoinositide breakdown, the first peak of carbachol (1 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2). The second peak was found to be related to [32P]PIP2 breakdown. Carbachol stimulated the release of [3H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [3H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [3H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-microM)-induced release of [3H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.     

Substance P-induced diacylglycerol formation in rat parotid acinar cells.

The mechanisms underlying the ability of substance P, to stimulate the sn-1,2-diacylglycerol (DAG) formation were studied using rat parotid acinar cells. During a 60 s stimulation, 1 microM substance P caused a rapid rise in DAG accumulation at 5 s, whereas a low (0.1 microM) concentration of agonist did not. During long term stimulation for 30 min, DAG accumulation induced by 1 microM substance P reached near maximal levels at 5 min and remained elevated for at least 20 min. In contrast, DAG formation induced by 0.1 microM substance P exhibited a peak at 5 min, gradually declined and returned to near basal levels at 30 min. Furthermore, DAG accumulation in response to substance P at 5 and 20 min increased in a dose-dependent manner. The breakdown of both [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2) stimulated by 1 microM substance P significantly increased from 5 to 20 min and returned to basal levels by 30 min; however, the breakdown of [32P]PIP2 was greater than that of [32P]PIP. At a low concentration of substance P, [32P]PIP2 breakdown reached maximal levels at 5 min followed by a progressive decrease and returned to basal levels at 30 min, whereas the breakdown of [32P]PIP reached maximal levels at 5 min and returned to near basal levels at 10 min. Both concentrations of substance P caused some [32P]phosphatidylinositol breakdown at 5 min. Changes in [3H]inositol trisphosphate induced by substance P were similar to those in [32P]PIP2. In addition, substance P (1 microM) did not stimulate the release of [3H]choline or [3H]ethanolamine metabolites into the medium. Substance P-induced DAG formation was not inhibited by staurosporine, a protein kinase C inhibitor. These results suggest that DAG formation caused by substance P is closely associated with the hydrolysis of phosphatidylinositides but not that of phosphatidylcholine or phosphatidylethanolamine, and is not regulated by protein kinase C-dependent mechanism(s).     

Effect of the tridecamer of compound 48/80, a Ca2+-dependent histamine releaser,on phospholipid metabolism during the early stage of histamine release from rat mast cells

When the tridecamer component of compound 48/80 (Fraction D, Fr. D), a Ca²⁺-dependent histamine releaser, was incubated with rat mast cells that had been prelabeled with [³²P]phosphate, [³H]inositol or [³H]glycerol, it induced a rapid decrease in [³²P]phosphatidylinositol-4,5-bisphosphate (PIP₂) followed by increases of [³H]inositol-1,4,5-triphosphate (Ins P₃) and [³H]diacylglycerol during the 10 sec prior to detectable histamine release. Fr.D-induced changes of the metabolism of these compounds occurred even in the absence of Ca²⁺, but to a lesser extent than in the presence of Ca²⁺. In contrast, the accumulation of [³H]arachidonic acid into phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidic acid (PA) in [³H]arachidonic acid-prelabeled mast cells was Ca²⁺-dependently stimulated by Fr.D with a concomitant decrease in [³H]phophatidylethanolamine (PE). These Ca²⁺-dependent changes in PC and PE were not observed in mast cells preloaded with [³²P]phosphate, while [³²P]PI and [³²P]PA increased Ca²⁺ independently. Fr.D also increased ⁴⁵Ca²⁺ uptake by mast cells within 5 sec after the stimulation. These results indicate that Fr.D binding to mast cell Ca²⁺ independently induces rapid changes of PI cycle-related metabolism of plasma membrane components, while it also induces Ca²⁺-dependent accumulation of arachidonic acid into PC, PI and PA in association with the decrease of PE, which may be important during the latent period prior to the Ca²⁺-dependent release of histamine from Fr.D-stimulated mast cells.     

Angiotensin II stimulation of phospholipase D in rat renal mesangial cells is mediated by the AT1 receptor subtype

Angiotensin II stimulated a biphasic 1,2-diacylglycerol formation in [³H]arachidonic acid-labelled mesangial cells. In contrast, in cells labelled with [³H]myristic acid, a tracer that preferentially marks phosphatidylcholine, angiotensin II induced a delayed monophasic production of 1,2-diacylglycerol. This delayed peak of 1,2-diacylglycerol generation was associated with a concomitant increase in choline formation, suggesting that stimulation of mesangial cells with angiotensin II causes a phospholipase D-mediated phosphatidylcholine hydrolysis. This conclusion is supported by the observation that angiotensin II stimulated the accumulation of phosphatidylethanol, when ethanol was added to mesangial cells. The production of choline and phosphatidylethanol stimulated by angiotensin II was completely blocked by the angiotensin II AT₁ receptor-selective antagonist DuP 753 with an IC₅₀ value of 8 nM, but not by the angiotensin II AT₂ receptor selective ligand CGP 42112A. Furthermore, angiotensin(1–7) and angiotensin(1–6) had only weak effects on choline generation. These data clearly indicate that angiotensin II AT₁ receptors trigger phospholipase D-mediated phosphatidylcholine hydrolysis in rat mesangial cells.     

Mechanism of the enrichment of phosphatidylcholine in liver accompanying enzyme induction by phenobarbital

Summary The mechanism of the increase of phosphatidylcholine in liver, accompanying enzyme induction by phenobarbital, has been studied in rats. Using radioactively labeled precursors, the two main pathways of phosphatidylcholine biosynthesis-the CDP-choline pathway and the methylation of phosphatidylethanolamine-were analyzed after pretreatment with 4 doses of phenobarbital (80 mg/kg) on 3 consecutive days.After i.v. injection of choline [Me-³H], choline [Me-¹⁴C] or NaH₂[³²P]O₄ the specific radioactivity (sp. act.) of phosphatidylcholine (dpm/nmol) was decreased by 60%, and after methionine [Me-³H] or ethanolamine [1.2-¹⁴C] by 40% compared to control rats.These changes are partly due to the increased concentration of phosphatidylcholine and phosphatidyl-ethanolamine, causing the incorporated precursors to dilute, and partly to a secondary effect which leads to a reduction of the sp. act. of free choline in pretreated animals.The concentration of glycerylphosphorylcholine, one of the metabolites of phosphatidylcholine catabolism, was also diminished by almost 50%.From these results it may be concluded that the increase of phosphatidylcholine is due to a retardation of its breakdown rather than to an increase of its synthesis.     

Protein kinase C-dependent diacylglycerol formation is mediated via Ca2+/calmodulin in parotid cells.

The kinetics of carbachol-induced sn-1,2-diacylglycerol (DAG) formation and the underlying mechanism(s) involved in parotid acinar cells were investigated. Supramaximal concentrations of carbachol for amylase secretion (10 microM) caused a transient rise in DAG levels at 10 s. In contrast, this rapid rise was not elicited by 1 microM carbachol, which is the maximally effective concentration for amylase secretion. Carbachol (10 microM) also increased DAG levels linearly up to 20 min, which were sustained for up to a further 10 min. DAG formation stimulated by 1 microM carbachol was biphasic; the first peak was observed after 5 min and the second after 20 min. DAG formation induced by 0.01-0.1 microM carbachol was concentration-dependent and monophasic, peaking at 5 min. The second peak evoked by carbachol was partly inhibited by Ca2+ deprivation from the extracellular space, whereas the first peak was not. Similar results were obtained in experiments using Ca2+ antagonists such as verapamil and LaCl3. The protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine, and a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), significantly inhibited the second DAG peak produced by 1 microM carbachol, but did not alter the first peak. The degree of inhibition of the second peak by these antagonists was comparable. Furthermore, the inhibitory effect of staurosporine and W-7 was concentration-dependent. The A23187-induced accumulation of DAG also was abolished by both staurosporine and W-7. These data indicate that a protein kinase C-dependent mechanism(s) is involved in mediating the second DAG accumulation peak induced by 1 microM carbachol and is mainly regulated by the Ca(2+)-calmodulin complex.     

Mechanism of the contractile response to Platelet-Activating Factor (PAF) of the rat stomach fundus. II. PAF-induced phosphatidylinositol turnover and desensitization

• 1.1. Accumulation of [³H]-inositol phosphates (IPs) was slightly enhanced by PAF in a concentration-dependent manner, but the accumulation was very small as compared with that induced by carbachol.
• 2.2. The levels of [³²P]-phosphatidic acid which is transformed from diacylglycerol (DAG) were increased by treatment with PAF or with carbachol.
• 3.3. PAF-induced contraction was significantly reduced by treatment with phorbol 12-myristate 13-acetate (PMA).
• 4.4. These results suggest that while PAF slightly stimulates the turnover of phosphatidylinositol (PI) in the rat stomach fundus, this response may not be responsible for the PAF-induced contractile response, and that the desensitization induced by repeated application of PAF may be due to the activation of protein kinase C.

     

Effect of pentobarbital on regional brain phospholipid synthesis

Rats were given 45 mg/kg i.p. sodium pentobarbital 15 min prior to the intraventricular injection of 200 μCi [³²P]phosphoric acid and 50 μCi [³H]glycerol. The animals were sacrificed 1 hr later, subcellular fractions were prepared from four subcortical brain regions and phospholipids were extracted. Pentobarbital significantly increased the ratio of [³H]- and [³²P]-triphosphatidylinositol (TPI) to diphosphatidylinositol (DPI) in the microsomal but not synaptosomal fractions. The possible relationship of this change to nicotinic receptor activity is discussed. Pentobarbital specifically decreased ³²Pi but not [³H]glycerol incorporation into synaptosomal phosphatidylinositol (PI). Thus, pentobarbital induced the opposite of the “neurotransmitter effect” on PI turnover. Pentobarbital either decreased or had no effect on the incorporation of ³²Pi and [³H]glycerol into phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE).     

Lutein is a competitive inhibitor of cytosolic Ca²+-dependent phospholipase A₂

Objectives We have investigated the effect of lutein on phospholipase A₂ (PLA₂) isozymes. Methods We measured arachidonic acid release in [³H]arachidonic acid‐labelled Raw 264.7 cells and PLA₂ activity using 1‐palmitoyl‐2‐[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA‐PC) and 10‐pyrene phosphatidylcholine in vitro. Key findings Lutein suppressed the release of arachidonic acid and inhibited Raw 264.7 cell‐derived cytosolic Ca²⁺‐dependent PLA₂ (cPLA₂)‐induced hydrolysis of [¹⁴C]AA‐PC in a dose‐ and time‐dependent manner. In contrast, lutein did not affect secretory Ca²⁺‐dependent PLA₂ (sPLA₂)‐induced hydrolysis of [¹⁴C]AA‐PC. A Dixon plot showed that the inhibition by lutein on cPLA₂ appeared to be competitive with an inhibition constant, K_i, of 13.6 µm . Conclusions We suggest that lutein acted as a competitive inhibitor of cPLA₂ but did not affect sPLA₂.     

Effects of R(−)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588), a novel cognitive enhancer,on noradrenaline release in rat cerebral cortical slices

We investigated the effects of R(−)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588), a novel cognitive enhancer, on noradrenaline (NA) release from rat cerebral cortical slices in vitro. Addition of T-588 in an assay mixture stimulated [³H]NA release from prelabeled slices in the presence or absence of extracellular CaCl₂, and in the presence of the Ca²⁺/calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. T-588 stimulated NA release with a time lag of about l min, and the high level of release was maintained for at least 10 min, whereas maximal KCl-evoked NA release was observed within l min after the addition of KCl, and the effect declined subsequently. The effect of T-588 was reversible (pretreatment with T-588 showed no effect on NA release after two washes by centrifugation). We also compared the effects of T-588 and N-ethylmaleimide (NEM), a sulfhydryl alkylating agent known to stimulate neurotransmitter release in several types of cells. The addition of NEM stimulated NA release irreversibly from the slices in a Ca²⁺-independent manner, and the effect of NEM, but not that of T-588, was inhibited by the simultaneous addition of dithiothreitol, a sulfhydryl group reducing agent. The addition of T-588, which stimulated NA release by itself, inhibited the NA release by 0.6 mM NEM, although the effect of T-588 was additive in the presence of 0.2 mM NEM. These findings suggest that T-588 stimulates NA release from rat cerebral cortical slices in a Ca²⁺- and calmodulin-independent manner, possibly via an NEM-sensitive factor(s), although the mechanism of the effects of T-588 seems to be different from that of NEM.     

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction

In gallbladder smooth muscle, carbachol interacts with M₃ receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [³H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates and contraction were measured at various times (0–90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 g/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [³²P]ADP-ribosylation of a –40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates. The time course of [³H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [³H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [³H]inositol trisphosphates and on contraction were abolished by atropine. Pretreatment with pertussis toxin resulted in ADP-ribosylation of a 40/41 kDa protein from gallbladder cell membranes but did not affect the concentration-response or time course of carbachol-induced contraction. These results indicate that carbachol-induced contraction of gallbladder smooth muscle cells is accompanied by the activation of inositol phosphate turnover and does not involve a pertussis toxin-sensitive G-protein.This article is based in part on the doctoral thesis of Burkhard Mackensen at the Faculty of Medicine, University of Hamburg, Germany. Some of the results were presented at the meeting of the American Gastroenterological Association (AGA) in San Francisco 1992 (von Schrenck et al. 1992) Correspondence to: T. von Schrenck at the above address     

Increased formation of phosphatidic acid induced with vasopressin or Ca2+ ionophore A23187 in rat hepatocytes

The effects of vasopressin and Ca2+ ionophore A23187 on phospholipid metabolism were investigated in rat hepatocytes. Vasopressin stimulated the incorporation of [32P]Pi into phosphatidic acid within 2 min but then it returned to control level after 10 min. On the other hand, the stimulation of the incorporation of [32P]Pi into phosphatidylinositol continued with incubation times up to 20 min. The Ca2+ ionophore A23187 also increased the 32P-labeling in phosphatidic acid, although it had no effect on [32P]Pi incorporation into phosphatidylinositol. Concerning the incorporation of [3H]glycerol, vasopressin did not enhance its incorporation into phosphatidic acid and phosphatidylinositol. The Ca2+ ionophore A23187 increased the incorporation into phosphatidic acid without significant effects on that into phosphatidylinositol. In the hepatocytes prelabeled with [3H]arachidonic acid, stimulated degradation of phosphatidylinositol with the addition of vasopressin and resultant formation of phosphatidic acid were observed within 5 min. The transient accumulation of diacylglycerol, the product of phosphatidylinositol hydrolysis, also occurred within 5 min with vasopressin. On the other hand, with the Ca2+ inophore A23187, stimulated degradation of triacylglycerol to diacylglycerol and the consequent formation of phosphatidic acid were observed. The Ca2+ ionophore A23187 caused a significant release of free [3H]arachidonic acid, although vasopressin had no effect.     

Drug-protein conjugates—II: An investigation of the irreversible binding and metabolism of 17α-ethinyl estradiol in vivo

Irreversible binding of radiolabelled material derived from [6,7-³H]17α-ethinyl estradiol ([³H]EE₂) to rat liver microsomal and soluble proteins occurred in vivo. It was measured by exhaustive solvent extraction and equilibrium dialysis. Three hours after administration of 5.0 μg kg^?1, 0.27 ± 0.14% (mean ± S.D., N = 4) of the dose was irreversibly bound to hepatic microsomes and 0.24 ± 0.16% to soluble protein. Induction of hepatic microsomal cytochrome P-488 and cytochrome P-450 by β-naphthoflavone (BNF) and phenobarbitone, respectively, did not significantly alter the irreversible binding of [³H]EE₂. Although enzyme induction did not affect the extent of binding, treatment with BNF, but not phenobarbitone, altered the quantitative pattern of the sulphated biliary metabolites of [³H]EE₂. The sulphated metabolites excreted by BNF-dosed rats comprised a significantly (P < 0.005) greater proportion of 2-hydroxyEE₂ than those excreted by vehicle-dosed controls. The increase was principally due to a decline in the proportion of 2-methoxyEE₂. It was suspected that the biliary metabolites of [³H]EE₂ insusceptible to hydrolysis might include conjugates formed by reactions between thiols and the reactive metabolite(s) of [³H]EE₂. Therefore l-[³⁵S]cysteine was administered prior to EE₂ in an attempt to label on or more of them. However, Except for an ³⁵S-labelled component also excreted by rats given only l-[³⁵S]cysteine, none of the sulphur-labelled biliary components co-eluted with a major [³H]EE₂ metabolite.     

Morinda citrifolia Inhibits Both Cytosolic Ca2+-dependent Phospholipase A2 and Secretory Ca2+-dependent Phospholipase A2

This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase A₂ (PLA₂) isozyme. PLA₂ activity was measured using various PLA₂ substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA-PC), and [³H]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [₃H]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited cPLA₂/sPLA₂-induced hydrolysis of [¹⁴C]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on cPLA₂ and sPLA₂ appeared to be competitive with inhibition constants (K_i) of 3.7µg/ml and 12.6µg/ml, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both Ca²⁺-dependent PLA₂ such as, cPLA₂ and sPLA₂. Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to Ca²⁺-dependent PLA₂ inhibition.     

Effect of temperature on muscarinic cholinoceptor-mediated phosphoinositide metabolism and tension generation in bovine tracheal smooth muscle

The effect of decreased temperature on phosphoinositide metabolism was studied in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM) by investigating the consequences of cooling on muscarinic-cholinoceptor-mediated [³H]inositol phosphate ([³H]InsP) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) accumulation, basal phosphoinositidase C (PIC) activity and airways smooth muscle (ASM) tone. Cooling of [³H]Ins labelled BTSM slices from 37°C to 27°C for 20 min prior to the addition of agonist caused a substantial (73.0±2.5%) inhibition of carbachol (100 M, 30 min)-stimulated [³H]InsP accumulation compared to values measured at 37°C. The degree of inhibition of [³H]InsP accumulation was similar at all agonist time points (2–30 min) studied. In parallel experiments, cooling of unlabelled BTSM slices from 37°C to 27°C resulted in a 34% reduction in basal Ins(1,4,5)P₃ mass (37°C, 13.1±0.6 pmol mg^– protein; 27°C, 8.9±0.9 pmol mg^–1 protein; P<0.02) and markedly attenuated carbachol (100 M)-stimulated increases in Ins(1,4,5)P₃ accumulation. Basal PIC activity in the soluble fraction of BTSM homogenates, measured using a [³H]phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) /deoxycholate assay system, was also significantly lower at 27°C compared to 37°C (initial velocities of PtdIns(4,5)P₂ hydrolysis of 853±167 (37°C) and 418±119 (27°C) pmol min^–1 ml^–1 (1/400 diluted) BTSM cytosol; p<0.02). Cooling of BTSM strips from 37°C to 27°C for 20 min affected neither the lag period prior to the onset of contraction, the rate of force development, nor the final magnitude of the tension generated by carbachol (100 M). However, a significant attenuation of the contractile response by cooling to 27°C was observed using a submaximal (EC₂₀) concentration of carbachol. Also, the contractile response to 1 mM McN-A-343, a partial agonist at M₃-cholinoceptors was significantly attenuated at 27°C with mean increases in the lag time and the t_1/2 to achieve maximal contraction of 558% and 369% respectively and a mean decrease in the maximum force generated of 37%. Despite previous reports indicating that cooling can enhance agonist-stimulated [³H]InsP₃ accumulation in certain tissues, modest degrees of cooling clearly inhibit basal and carbachol-stimulated phosphoinositide hydrolysis in bovine tracheal smooth muscle and reduces the muscarinic receptor reserve in tracheal smooth muscle contraction.     

Mechanisms for the increase in electrically stimulated [3H]norepinephrine release from rat cortical slices by N-(n-propyl)-N-(4-pyridinyl)-1H-indol-1-amine

N-(n-propyl)-N-(4-pyridinyl)-1H-indol-1-amine (HP 749) is currently in clinical trials for the treatment of Alzheimer's disease (AD). While HP 749 has many pharmacological properties, the biochemical basis for its efficacy in animal models for AD remains unexplained. To this end, we have investigated some biochemical properties of HP 749 as they relate to its effect on electrically stimulated [³H]norepinephrine (NE) release. HP 749 was found to inhibit both [³H]NE uptake and [³H]yohimbine binding to cortical μ₂-adrenergic receptors. Consistent with this profile, HP 749 (1 and 10 μM) enhanced electrically stimulated release of [³H]NE from rat cortical slices. Both clonidine (1 μM) and nomifensine (10 μM) inhibited the effect of HP 749 (1 μM). The enhancement of [³H]NE release produced by the μ₂ adrenergic antagonist, idazoxan (0.1 μM), was completely reversed by the μ₂ agonist, clonidine (1 μM), but was not affected by the NE uptake inhibitor, nomifensine (10 μM). These results indicate that the HP 749 enhancement of electrically stimulated [³H]NE release is due, at least in part, to a combination of presynaptic μ₂-adrenergic receptor antagonism and NE reuptake blockade. These mechanisms may contribute to some of the adrenergic effects of HP 749.     

Effect of soman on N-methyl-d-aspartate-stimulated [3H]norepinephrine release from rat cortical slices

Effects of soman on N-methyl-d-aspartate (NMDA) evoked [³H]norepinephrine (NE) release were examined in rat brain cortical slices. NMDA increased [³H]NE release in a concentration-dependent manner. Soman could inhibit the increase evoked by NMDA, but carbachol, an agonist of cholinergic receptor, could potentiate the increase evoked by NMDA. Atropine (a selective muscarinic antagonist) attenuated the release of [³H]NE induced by NMDA in the presence of carbachol or acetylcholine (ACh), but had no effect on the release of [³H]NE induced by NMDA alone. Both d-tubocurarine (an antagonist of nicotinic receptor) and atropine had no effect on the release of [³H]NE induced by NMDA in the presence of soman. These results suggested that soman has a direct action at non-cholinergic sites, probably at NMDA receptors.     

Effect of acute ethanol or acetaldehyde adminstration on the uptake,release, metabolism and turnover rate of norepinephrine in rat brain

Administration of acetaldehyde to rats following an intracisternal injection of [³H]norepinephrine (NE) produced a decrease in brain endogenous NE with a concurrent increase in disappearance rate of [³H]NE. In contrast, acute ethanol administration appears to decrease both the rate of disappearance of [³H]NE and endogenous NE in brain. The pattern of NE metabolites in the brain was changed by acetaldehyde but not by ethanol. In experiments where ethanol was given prior to i.e. injection of [³H]NE, a small decrease in retention of [³H]NE observed at 15 min was followed by an increase at 90 min exposure to [³H]NE. There was a non-significant increase in [³H]normetanephrine formation at 15 min. These results suggest ethanol has a dual action on NE, initially increasing the release of NE and at the same time decreasing the neuronal uptake which may decrease the turnover rate of NE. However, acetaldehyde affects only the release of NE, thus increasing its turnover rate in the brain.     

Metabolism of prostaglandin E2 in the isolated perfused kidney of the rabbit

In the Krebs-perfused rabbit isolated kidney, [³H]PGE₂ (5 μCi, 165 Ci/mmole) was infused intra-arterially for 5 min; venous and urinary effluents were collected every 2 min for 20 min. Efflux of radioactive material peaked at 8 min and declined thereafter. The kidney retained 35% of the infused ³H. Samples were extracted for acidic lipids; PGE₂, PGF_2α and metabolites were separated by TLC and quantified by a radiometric method. Efflux of [³H]PGF_2α into urinary and venous outflows increased progressively over the first 12 min and then plateaued for the remaining 4 min. By 12 min, conversion of [³H]PGE₂ to [³H]PGF_2α was 70 and 80% as determined by radiolabeled products recovered in the urinary and venous effluents respectively. Estimates of total conversion of [³H]PGE₂ to [³H]PGF_2α were 62 and 52% of the radiolabeled material exiting in the urinary and venous effluents respectively. The 15-keto and 13,14-dihydro-15-keto metabolites of [³H]PGF_2α appeared in the urine but were not found in the venous outflow. We conclude that PGE-9-ketoreductase (PGE-9KRD) activity is high in the rabbit isolated perfused kidney. Further, the extent of conversion of PGE₂ to PGF_2α and metabolism of newly formed PGF_2α may differ within the vascular and tubular compartments of the kidney. PGE-9KRD activity may be important in the regulation of renal vascular tone, compliance of veins, and salt and water balance.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.