Expression of the Acc1 Gene-Encoded Acetyl-Coenzyme A Carboxylase in Developing Maize (Zea mays L.) Kernels.

A mutation (Acc1-S2) in the structural gene for maize (Zea mays L.) acetyl-coenzyme A carboxylase (ACCase) that significantly reduces sethoxydim inhibition of leaf ACCase activity was used to investigate the gene-enzyme relationship regulating ACCase activity during oil deposition in developing kernels. Mutant embryo and endosperm ACCase activities were more than 600-fold less sensitive to sethoxydim inhibition than ACCase in wild-type kernel tissues. Moreover, in vitro cultured mutant kernels developed normally in the presence of sethoxydim concentrations that inhibited wild-type kernel development. The results indicate that the Acc1-encoded ACCase accounts for the majority of ACCase activity in developing maize kernels, suggesting that Acc1-encoded ACCase functions not only during membrane biogenesis in leaves but is also the predominant form of ACCase involved in storage lipid biosynthesis in maize embryos.

Thiol methylation pharmacogenetics: heritability of human erythrocyte thiol methyltransferase activity.

Thiol methylation of aliphatic sulfhydryl drugs is catalyzed by thiol methyltransferase (TMT), an enzyme activity that can be measured in the human erythrocyte (RBC) membrane. As a first step toward determining the possible role of inheritance in the regulation of individual variations in the S-methylation of drugs in man, the heritability of human RBC membrane TMT activity was determined. RBC TMT activity was measured in blood samples from 231 first-degree relatives in 47 randomly selected families. The frequency distribution of enzyme activities was unimodal, with a fivefold variation within +/- 2 SDs. RBC TMT activity did not correlate with either age or sex. Heritability in the "narrow" sense (h2) was estimated by comparing correlations of RBC TMT activities in first-degree relatives with theoretical values expected for a trait under total additive genetic control. The correlation between RBC TMT activities in mothers and fathers in these families was only 0.04, a finding that made shared environment a less likely explanation for significant correlations among other family members. However, sibling-sibling (S-S), parent-offspring (P-O), and midparent (average of two parental values)-offspring (M-O) correlations were 0.49, 0.49, and 0.69.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative actions of synthetic omega-grammotoxin SIA and synthetic omega-Aga-IVA on neuronal calcium entry and evoked release of neurotransmitters in vitro and in vivo.

The effects of synthetic omega-grammotoxin SIA (omega-GsTxSIA) and synthetic omega-Aga-IVA were tested in in vitro and in vivo neurochemical assays that are reflective of voltage-sensitive calcium channel function. Synthetic omega-GsTx SIA inhibited K(+)-evoked rat and chick synaptosomal 45Ca2+ flux, K(+)-evoked release of [3H]D-aspartate and [3H]norepinephrine from rat hippocampal brain slices and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices with potency values that were comparable to those found previously with omega-GsTx SIA purified from the venom of the tarantula spider Grammostola spatulata. These results indicate that trace contaminants do not account for the pharmacology of purified omega-GsTx SIA. omega-GsTx SIA caused a complete inhibition of rat synaptosomal 45Ca2+ flux and hippocampal slice [3H]D-aspartate release, whereas omega-Aga-IVA caused a maximal inhibition of approx 75%. omega-GsTx SIA and omega-Aga-IVA caused an identical partial inhibition of K(+)-evoked increases of intracellular calcium in cortical neurons in primary culture. The addition of nitrendipine to either omega-GsTx SIA or omega-Aga-IVA resulted in an additive and virtually complete inhibition of the cortical neuron intracellular calcium response. In in vivo microdialysis studies, the K(+)-evoked release of glutamate from hippocampus of awake freely moving rats was inhibited with the following rank order of potency: omega-conotoxin GVIA > omega-GsTx SIA > omega-Aga-IVA. Complete inhibition of K(+)-evoked hippocampal glutamate release was observed with 300 nM omega-conotoxin GVIA and 3 microM omega-GsTx SIA. In urethane anesthetized rats, omega-CgTx GVIA caused a partial inhibition, whereas omega-GsTx SIA caused a concentration-dependent and complete inhibition, of basal serotonin release in the hippocampus. Therefore, omega-GsTx SIA was shown to inhibit responses that are sensitive to omega-conotoxin GVIA, omega-Aga-IVA and omega-conotoxin MVIIC, consistent with the notion that omega-GsTx SIA inhibits N-, P- and Q-type high threshold voltage-sensitive calcium channels.

Evolution of a series of peptidoleukotriene antagonists: synthesis and structure/activity relationships of 1,3,5-substituted indoles and indazoles.

1,3,5-Substituted indoles and indazoles have been studied as receptor antagonists of the peptidoleukotrienes. The best of these compounds generally had a methyl group at the N1 position, a [(cyclopentyloxy)carbonyl]amino or 2-cyclopentylacetamido or N'-cyclopentylureido group at the C-5 position, and an arylsulfonyl amide group as part of the acidic chain at the C-3 position of the ring. Such compounds had in vitro dissociation constants (KB) in the range 10(-9) - 10(-11) M on guinea pig trachea against LTE4 as agonist and inhibition constants (Ki) less than or equal to 10(-9) M on guinea pig parenchymal membranes against [3H]LTD4. A number of compounds were orally effective at doses less than or equal to 1 mg/kg in blocking LTD4-induced "dyspnea" in guinea pigs. Compound 45 [N-[4-[[5-[[(cyclopentyloxy)carbonyl]-amino]-1-methylindol-3- yl]methyl]-3-methoxybenzoyl]-2-methylbenzenesulfonamide, ICI 204,219; pKB = 9.67 +/- 0.13, Ki = 0.3 +/- 0.03 nM, po ED50 = 0.3 mg/kg] is currently under clinical investigation for asthma. In the indole series, certain alkylsulfonyl amides possessing a 3-cyanobenzyl substituent at the N-1 position (60, 61) were produced that had KB less than or equal to 10(-9) M on guinea pig trachea.

Individual variations of prostanoid agonist responses in rabbit aorta: evidence for the independent regulation of prostanoid receptor subtypes.

1 The frequency and selectivity of individual variations of prostanoid agonist responses in aortic strips from a population of male rabbits was studied. Three levels of responsiveness to the thromboxane mimetic U46619 occurred: responders (R), intermediate responders (IR), and non-responders (NR). R could be subdivided into R1 and R2 based on an enhanced potency of prostaglandin F2 alpha (PGF2 alpha) in R2. In the total population (n = 92), the phenotype frequency was: R, 69%; IR, 11%; and NR, 20%. In a subgroup of this population in which R1 and R2 phenotypes were determined (n = 63), the phenotype frequency was: R1, 54%; R2, 19%; IR, 6%; and NR, 21%. 2 The four rabbit aorta phenotypes, R1, R2, IR, and NR, were characterized with respect to the rank orders of prostanoid agonist potency, agonist intrinsic activities, and the effects of the thromboxane receptor antagonist SQ29548. The rank order of prostanoid agonist potency was U46619 greater than PGF2 alpha greater than PGE2 in R1 and R2, and PGF2 alpha greater than or equal to PGE2 greater than U46619 in IR and NR. For each prostanoid agonist, the intrinsic activity was highest in R (R1 congruent to R2), intermediate in IR, and lowest in NR. In R1, SQ29548 inhibited all prostanoid agonist responses equally. The contractile effects of PGF2 alpha and PGE2 were partially resistant to inhibition by SQ29548 in R2. Prostanoid agonist responses were not inhibited by SQ29548 in IR. 3 The potency of histamine was equivalent in R1, R2, IR, and NR. 4 It is concluded that there are individual variations in the functional expression of thromboxane receptor sensitivity, i.e., prostanoid agonist responses inhibited by SQ29548. Also, there are individual variations in the functional expression of the sensitivity of a non-thromboxane receptor or receptors, i.e., prostanoid agonist responses not inhibited by SQ29548. It has been proposed by others that prostanoid receptors be termed P-receptors and that the prostanoid agonist to which they are most sensitive be indicated by a preceding letter, e.g., TP- for thromboxane receptor and FP- for PGF2 alpha-selective receptor. Accordingly, we proposed a working hypothesis that suggests the four phenotypes could result from the independent regulation of the functional expression of TP- and FP-receptor subtypes with (a) R2 containing both the TP- and FP-receptor subtypes in a fully functional state; (b) R1 containing only the functional TP-receptor; (c) IR containing only the functional FP-receptor; and (d) NR containing only a low efficacy FP-receptor system. 5 The mechanisms underlying the observed individual variations are unknown but could include changes in receptor number or affinity, changes in receptor-effector coupling, changes in a second messenger system, or changes in tissue degradative or uptake processes. Further study is needed to differentiate between these possibilities.

Inhibition of N-methyl-D-aspartate- and kainic acid-induced neurotransmitter release by omega-conotoxin GVIA.

1. The role of voltage-sensitive calcium channels (VSCC) in N-methyl-D-aspartate (NMDA)- and kainic acid (KA)-evoked neurotransmitter release from rat cortical and hippocampal brain slices was evaluated by determining the effects of omega-conotoxin GVIA, an inhibitor of neuronal L- and N-type VSCC, and PN 200-110, a selective inhibitor of L-type VSCC. 2. Selective antagonists of the NMDA receptor ionophore complex, Mg2+, CPP and MK-801, inhibited NMDA- but not KA-evoked release of [3H]-noradrenaline from hippocampal and cortical brain slices. This suggests that cortical and hippocampal receptors are similar and that NMDA and KA act at distinct excitatory amino acid receptor subtypes. 3. [3H]-noradrenaline release induced by both NMDA and KA was similarly inhibited (approximately 30%) by omega-conotoxin GVIA. In contrast, PN 200-110 had no significant effect, although there was a tendency towards inhibition. 4. The results suggest that although NMDA- and KA-receptors are pharmacologically distinct, the N-type, but not the L-type, VSCC plays a small but significant role in neurotransmitter release induced by both NMDA and KA. It remains to be determined whether the N-type VSCC are involved in the physiological and/or pathological manifestations of excitatory amino acid receptor stimulation.

Differential inhibition of neuronal calcium entry and [3H]-D-aspartate release by the quaternary derivatives of verapamil and emopamil.

1. Verapamil and emopamil are structurally related phenylalkylamine calcium channel/5-HT2 receptor antagonists that differ in their anti-ischaemic properties in experimental studies. The quaternary ammonium derivatives of these compounds were prepared and tested in assays of neuronal voltage-sensitive calcium channel (VSCC) function to determine whether the compounds act at intra- or extracellular sites. 2. The compounds were tested in K(+)-evoked: (1) rat brain synaptosomal 45Ca2+ influx, (2) release of [3H]-D-aspartate from rat hippocampal brain slices and (3) increase of intracellular calcium in rat cortical neurones in primary culture. 3. Verapamil, emopamil and the emopamil quaternary derivative caused concentration-dependent and comparable (IC50 values approximately 30 microM) inhibition of synaptosomal 45Ca2+ influx and [3H]-D-aspartate release. The verapamil quaternary derivative was considerably less active in these assays (IC50 > 300 microM). 4. The evoked increase of intracellular calcium in cortical neurones was inhibited with the following rank order of potency (IC50 value, microM): emopamil (3.6) > verapamil (17) > emopamil quaternary derivative (38) > verapamil quaternary derivative (200). 5. The results suggest that verapamil and emopamil inhibit nerve terminal VSCC function (synaptosomal 45Ca2+ influx and [3H]-D-aspartate release) by acting at distinct intracellular and extracellular sites, respectively. Verapamil and emopamil may inhibit cell body VSCC function (evoked increase of intracellular calcium in neocortical neurones) by acting at both intracellular and extracellular sites. 6. The different 'sidedness' of action of emopamil and verapamil on nerve terminal VSCC function and/or the preferential inhibition of cell body VSCC function by emopamil may at least partially explain the relatively greater neuroprotective efficacy of emopamil in experimental models of ischaemia.

Thiopurine methyltransferase: mouse kidney and liver assay conditions, biochemical properties and strain variation.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of aromatic and heterocyclic thiol compounds including drugs such as 6-mercaptopurine (6-MP) and 6-thioguanine. In humans, the level of TPMT activity is inherited in a monogenic fashion. It would be important to develop an experimental animal model in which the genetic regulation of TPMT could be studied. Therefore, TPMT activity was measured in kidney and liver homogenates from A/J inbred mice. Apparent Michaelis (Km) constants for the two cosubstrates for the reaction, 6-MP and S-adenosyl-L-methionine (Ado-Met), in mouse kidney were 7.0 X 10(-4) M and 2.4 X 10(-6) M respectively. Apparent Km constants for 6-MP and Ado-Met in mouse liver were 5.4 X 10(-4) M and 2.1 X 10(-6) M respectively. The pH optimum for the reaction was 6.7 in both tissues, and over 95% of the TPMT activity in both mouse liver and kidney was "soluble" after centrifugation at 100,000 g for 1 hr. 3,4-Dimethoxy-5-hydroxybenzoic acid, an inhibitor of human kidney TPMT, decreased mouse kidney and liver enzyme activities by more than 95% at a concentration of 1 mM. TPMT activities were then measured in liver and kidney tissue from nine additional inbred strains of mice aged 7-8 weeks. Six of the nine inbred strains had TPMT activities very similar to those found in A/J animals. However, three strains, the C57BL/6J, C57BL/6ByJ and AKR/J, had significantly lower levels of activity in both liver and kidney than did any of the seven other strains. Liver TPMT activities in these three strains were only 23-32% of the average activity in A/J mouse liver. Kidney enzyme activities in the same three strains averaged 49-62% of the average activity in A/J mouse kidneys. These striking differences in TPMT activity among inbred mouse strains will make it possible to test the hypothesis that inheritance regulates variations in TPMT activity in this experimental animal.

Effects of stimulus intensity on the inhibition by omega-conotoxin GVIA and neomycin of K(+_-evoked [3H]norepinephrine release from hippocampal brain slices and synaptosomal calcium influx.

The effects of various K+ concentrations on the inhibition of [3H]norepinephrine release from rat hippocampal brain slices and evoked synaptosomal 45Ca2+ influx by omega-conotoxin GVIA (omega-CgTx) and neomycin were examined. K+ (15-75 mM) caused a concentration-dependent release of [3H]norepinephrine that was greater than 90% dependent on extracellular calcium. The ability of omega-CgTx to inhibit [3H]norepinephrine release was optimal at 25 mM K+ and was reduced substantially at higher concentrations of K+. omega-CgTx maximally inhibited [3H]norepinephrine release by 49% (15 mM K+), 58% (25 mM K+), 22% (50 mM K+), and 12% (75 mM K+). In contrast, neomycin caused a concentration-dependent and virtually complete inhibition of [3H]norepinephrine release at all concentrations of K+, with IC50 values of 210 microM (15 mM K+), 150 microM (25 mM K+), 450 microM (50 mM K+), and 1500 microM (75 mM K+). omega-CgTx (1 microM) had little effect (10% or less inhibition) on hippocampal synaptosomal 45Ca2+ influx at any concentration of K+, whereas 3 mM neomycin caused at least 75% inhibition of 45Ca2+ influx, with the largest inhibition (96%) occurring at 25 mM K+. The results suggest that increasing stimulus intensity decreases the contribution of N-type voltage-sensitive calcium channels (VSCC) in mediating K(+)-evoked release of [3H]norepinephrine. The comparative absence of omega-CgTx-sensitive synaptosomal 45Ca(2+)-influx sites suggests that N-type calcium channels are a small subset of channels in rat hippocampal synaptosomes. The demonstration that neomycin can inhibit omega-CgTx-sensitive and -insensitive neurotransmitter release and calcium influx suggests that neomycin may block N-type VSCC as well as non-N-type VSCC.

Role of calcium in regulation of phosphoinositide signaling pathway.

Using primary neuronal cultures we have examined the role of extracellular Ca2+ in a receptor-regulated phosphoinositide turnover. We report that receptor (glutamic acid and acetylcholine)-activated phosphoinositide turnover requires the presence of extracellular Ca2+ (EC50 = 21.1 microM). The requirement for Ca2+ appears to be at an intracellular level and is highly selective for Ca2+. We also found that several inorganic and organic Ca2+ channel blockers, including La3+ and verapamil, inhibit phosphoinositide turnover. However, the pharmacological profile of these agents in this regard was distinct from their actions at the voltage-sensitive Ca2+ channels. To explain the above requirement for extracellular Ca2+ in agonist-stimulated phosphoinositide turnover and its sensitivity to Ca(2+)-channel blockers, we propose a hypothetical model suggesting that Ca2+, following IP-3-mediated mobilization, exerts a facilitatory action on the activity of receptor-phospholipase C complex. We further propose that in the absence of extracellular Ca2+ or in the presence of certain Ca(2+)-channel blockers, refilling of calciosomes is ineffectual or inhibited, causing its depletion and subsequent inactivation of agonist-stimulated phosphoinositide turnover.

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses.

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.

Differential sensitivity to lithium's reversal of amphetamine-induced open-field activity in two inbred strains of mice.

To determine whether genetic differences could contribute to the pharmacological sensitivity of lithium chloride (LiCl) to reverse amphetamine-associated changes in behavior C57BL/6nCrlBR and C3H/HenCrlBR male mice were tested for the ability of an acute dose of LiCl to reverse the locomotor enhancing effects of an acute dose of amphetamine. A series of experiments were conducted that compared dose response of LiCl, chamber lighting conditions, and chamber shape on amphetamine-induced activity in two strains of mice with different genetic backgrounds. Acute amphetamine (3 mg/kg) increased locomotor activity in C57BL/6nCrlBR mice and LiCl (1-4 mEq/kg) blocked this effect. LiCl-induced changes in baseline activity seen at high doses of LiCl were not seen for the low doses. The dark condition reduced time resting but chamber shape did not appear to alter results. In C3H/HenCrlBR mice, amphetamine did not significantly increase levels of activity but did decrease rearing behavior which suggests that genetic difference between C57BL/6nCrlBR and C3H/HenCrlBR mice may contribute to sensitivity to amphetamine. In sum, the ability of LiCl to reverse amphetamine-induced changes in locomotor activity in C57BL/6nCrlBR mice may provide a useful model to study genetic and pharmacological aspects of psychiatric illnesses such as bipolar disorder.

Inhibition of K(+)-evoked [3H]D-aspartate release and neuronal calcium influx by verapamil, diltiazem and dextromethorphan: evidence for non-L/non-N voltage-sensitive calcium channels.

The effects of inhibitors of voltage-sensitive calcium channels (VSCC) on K(+)-evoked [3H]D-aspartate release from rat hippocampal slices and the K(+)-evoked increase in intracellular calcium in neocortical neurons in primary culture were examined. K+ caused a concentration-dependent release of [3H]D-aspartate that was approximately 85% dependent on the presence of extracellular calcium. Neither the marine snail toxin, omega-conotoxin GVIA, nor the dihydropyridine VSCC antagonist, nitrendipine, had any effect on K(+)-evoked release of [3H]D-aspartate. omega-Conotoxin GVIA and nitrendipine caused a relatively small (20-30%) inhibition of K(+)-evoked increase in intracellular calcium in neocortical neurons in primary culture. This suggests that K(+)-evoked [3H]D-aspartate release is not dependent on L- or N-type VSCC, whereas K(+)-evoked neuronal calcium influx was only partially dependent on L- and N-type VSCC. Verapamil, dextromethorphan and diltiazem caused a concentration-dependent inhibition of K(+)-evoked release of [3H]D-aspartate with IC50 values of 30, 100 and 120 microM, respectively. The K(+)-evoked increase in intracellular calcium was inhibited with essentially the same rank order of potency, but with slightly greater potencies (IC50 values for verapamil, diltiazem and dextromethorphan were 20, 50 and 50 microM, respectively). At 300 microM, neither verapamil, diltiazem nor dextromethorphan inhibited [3H]D-aspartate release evoked by the calcium ionophore ionomycin, suggesting that these compounds are not acting intracellularly to inhibit the ability of free cytosolic calcium to evoke release.(ABSTRACT TRUNCATED AT 250 WORDS)

HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartate-evoked neurotransmitter release.

The role of endogenous glycine in supporting N-methyl-D-aspartate (NMDA)-evoked neurotransmitter release was investigated. HA-966 (1-hydroxy-3-aminopyrrolidone-2) inhibited NMDA-evoked release of [3H]norepinephrine from rat hippocampal brain slices, but was much less effective in inhibiting [3H]norepinephrine release evoked by kainic acid (KA). Glycine (1 mM) reversed the HA-966 (1 mM) antagonism of NMDA-evoked release of [3H]norepinephrine. Strychnine (10 microM) had no effect on the ability of glycine to reverse HA-966 antagonism of NMDA-evoked neurotransmitter release. Other amino acids were also capable of reversing the HA-966 antagonism of NMDA-evoked [3H]norepinephrine release with a rank order of potency: D-serine greater than or equal to glycine much greater than L-serine approximately beta-alanine. These same compounds inhibited strychnine-insensitive [3H]glycine binding to rat cortical membrane fragments with a rank order of potency: glycine greater than D-serine much greater than L-serine greater than or equal to beta-alanine. In addition, HA-966 inhibited [3H]glycine binding (IC50 = 8.5 microM). The results suggest that HA-966 antagonism of NMDA-evoked neurotransmitter release is due to the inhibition of endogenous glycine acting at a strychnine-insensitive modulatory glycine site associated with the NMDA receptor/ionophore complex.

Complete and reversible block by omega-grammotoxin SIA of glutamatergic synaptic transmission between cultured rat hippocampal neurons.

omega-Grammotoxin SIA (omega-GsTx SIA), a peptide isolated from tarantula venom, inhibits synaptosomal Ca2+ influx and neurotransmitter release, and blocks N-, P-, and Q-type voltage-gated Ca2+ channels. The whole-cell patch-clamp was used to record glutamatergic excitatory post-synaptic currents (EPSCs) evoked by extracellular stimulation of presynaptic neurons in primary rat hippocampal cultures. EPSCs displayed rapid kinetics and were blocked by CNQX. omega-Conotoxin (1 microM) GVIA inhibited EPSCs by 46%, while 30 nM and 1 microM omega-agatoxin IVA produced 12% and 69% inhibition, respectively, consistent with coupling of N-, P- and Q-type Ca2+ channels to glutamatergic synaptic transmission. omega-GsTx SIA (1 microM) rapidly, completely, and reversibly blocked glutamatergic EPSCs, but did not affect currents evoked by bath application of kainate. Thus, omega-GsTx SIA blocks glutamatergic synaptic transmission by blocking presynaptic voltage-gated Ca2+ channels. omega-GsTx SIA is the only agent that blocks selectively and reversibly the Ca2+ channels coupled to glutamate release. omega-GsTx SIA provides a unique and powerful tool for experiments requiring recovery of function following presynaptic block of synaptic transmission.

Inhibition of presynaptic alpha-2-adrenoceptor and opioid receptor agonist responses in the rat vas deferens by chronic imipramine treatment.

The effects of chronic imipramine administration on agonist responses in rat isolated smooth muscle preparations were investigated. The administration of 20 mg/kg imipramine two times a day for 4 and 11 days resulted in an equivalent subsensitivity (approximately 8-fold) of clonidine-induced inhibition of electrically evoked contractions in the rat vas deferens (presynaptic alpha 2-adrenoceptor response). Imipramine (4 days) resulted in a marked inhibition of the ability of [D-Ala2, D-Leu5] enkephalin to decrease electrically evoked contractions of the vas deferens (presynaptic opioid receptor response) but did not significantly affect the carbachol-induced increase in electrically evoked contractions (muscarinic receptor response). In the absence of cocaine the contractile effects of norepinephrine and tyramine in the vas deferens were, respectively, potentiated and inhibited, following imipramine (4 days), suggesting a decrease in the activity of the neuronal uptake mechanism. When determined in the presence of cocaine, the potency of the postsynaptic effects of norepinephrine in the vas deferens (alpha 1-adrenoceptor response) was not significantly altered by imipramine (4 days). With regard to other postsynaptic receptors, imipramine (4 days) decreased slightly the potency of phenylephrine in the aorta (alpha 1-adrenoceptor response) and increased slightly the potency of carbachol in the trachea (muscarinic receptor response) and the potency of serotonin in the rat aorta (5HT2-receptor response). Thus, chronic imipramine administration decreased the potency of presynaptic alpha 2- and opioid agonist responses in the vas deferens but caused very little or no changes in the potencies of agonists at postsynaptic sites.

Correlation of low and high affinity thiol methyltransferase and phenol methyltransferase activities in human erythrocyte membranes.

Human red blood cell (RBC) membranes have been reported to contain both high and low affinity 'forms' of the drug metabolizing enzyme thiol methyltransferase (TMT). The biochemical characteristics of the two 'forms' of human RBC TMT were compared. Apparent Km constants of the high affinity activity for 2-mercaptoethanol and S-adenosyl-L-methionine, cosubstrates for the TMT reaction, were 0.38 mumol/l and 2.6 mumol/l, respectively. These constants may be compared with values of 20 mmol/l and 43 mumol/l, respectively, previously reported for the low affinity form of RBC TMT activity. The properties and regulation of the two forms of TMT were then compared with each other and also with those of two 'control' enzymes, phenol methyltransferase (PMT) and beta-glucuronidase. When high and low affinity TMT, PMT and beta-glucuronidase activities were measured in RBC membranes from 22 individual subjects, there were highly significant correlations among all three methyltransferase activities (all r values greater than 0.95), but beta-glucuronidase activity did not correlate significantly with any of the methyltransferase activities (all r values less than 0.40). The thermal stabilities of the three methyltransferases were very similar. They were all inactivated approximately 50% by incubation at 48 degrees C for 15 min. beta-Glucuronidase activity was approximately 50% inactivated by incubation at 76 degrees C for 15 min. PMT and both TMT activities had similar subcellular distributions and similar responses to ions and to enzyme inhibitors. These results suggested that high and low affinity TMT and PMT activities might be catalyzed by the same enzyme. Alternatively, these three RBC membrane methyltransferase activities might be regulated in a parallel fashion.

Time use of stroke patients in three rehabilitation hospitals.

The active role of the patient in the rehabilitation hospital requires the efficient use of patient time. Previous work has found that such patients spend a relatively small portion of their day in treatment. In this study 63 stroke patients in three hospitals were observed for two work days each. Fifty observations were made during each day of the patient's location, activity and whom the patient was with. Treatment occupied an average of 31% of the day. Passive behavior took up 42% of patients' time. Discriminant analysis showed that patients in the hospital with a separate stroke unit spent more time in treatment. In spite of quantitative differences, the three hospitals had similar patterns of treatment activity and deployment of staff. Clinical norms for the treatment of stoke across the three hospitals resulted in similar treatment experiences for patients. A system of prospective payment which did not require specific billed treatment hours might allow more flexibility and efficiency in this type of specialty hospital.

The effect of varying carbachol concentration on the slope of Schild plots of selective beta-adrenoceptor antagonists in the carbachol-contracted guinea-pig trachea.

The effect of varying the level of smooth muscle tone induced by carbachol on the Schild analysis of atenolol (beta 1-selective) and ICI 118,551 (beta 2-selective) with salbutamol as agonist, on the guinea-pig tracheal preparation has been examined. When 10(-6) M carbachol was used to induce near-maximal smooth muscle tone, Schild plot slopes for atenolol and ICI 118,551 were less than 1. Slopes of Schild plots for both drugs were equivalent to 1 when 10(-7) M carbachol was used to produce approximately half-maximal smooth muscle tone. Depletion of neuronal noradrenaline by prior treatment with reserpine had no effect on the Schild analysis. Salbutamol produced maximal relaxation and was more potent when tone was induced with 10(-7) M carbachol, but was less effective at 10(-6) M carbachol. Pretreatment with reserpine increased the potency of salbutamol at each concentration of carbachol. The results suggest that either the level of smooth muscle tone or an unknown effect associated with a high level of smooth muscle tone induced by carbachol may contribute to low slope values of Schild plots of selective beta-adrenoceptor antagonists in the carbachol-contracted guinea-pig trachea. The carbachol-contracted guinea-pig trachea can be used to determine theoretically valid pA2 values for selective beta-adrenoceptor antagonists as long as substantially less than a maximal level of smooth muscle tone is induced by carbachol.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).