Studies of mitochondrial calcium movements using chlorotetracycline

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 μM caused a transient fluorescence increase followed by an extensive decrease in fluorescence.

2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous ⁴⁵Ca. A positive correlation was observed between fluorescence and energized ⁴⁵Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chlorotetracycline fluorescence but an enhanced uptake of exogenous ⁴⁵Ca.

3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with ⁴⁵Ca. Endogenous ⁴⁵Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the ⁴⁵Ca content of the mitochondria or the fluorescence of the probe.

4. Mitochondria treated with antimycin A lost most of their endogenous ⁴⁵Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released ⁴⁵Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the ⁴⁵Ca previously released.

5. Following this energized uptake of ⁴⁵Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the ⁴⁵Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.     

Factors involved in the generation of tension during contraction to high potassium in the rat vas deferens

A large number of studies indicate that K⁺-induced contractions of smooth muscle depend on extracellular calcium. If these contractions depend exclusively on extracellular calcium then contractile responses to 140 mM K⁺, which are larger than the response to 35 mM K⁺, should be associated with a larger influx of ⁴⁵Ca. This is not the case in the vas deferens from reserpine pretreated rats. During a 2 min interval, ⁴⁵Ca influx induced by 140 mMK⁺ was identical to that produced by 35 mM K⁺. This suggests that a second mechanism may be involved in responses to high K⁺. Indeed, 140 mM K⁺ caused an approximately 300% increase above control in the formation of inositol trisphosphate (IP₃) in tissues prelabelled with ³H-myoionositol whereas 35 mM K⁺ did not increase IP₃. IP₃ is thought to cause the release of calcium from internal stores which is consistent with our finding of an increase in ⁴⁵Ca efflux into calcium-free medium from tissues prelabelled with ⁴⁵Ca and stimulated with 140 mM K⁺. Stimulation with 35 mM K⁺ did not influence ⁴⁵Ca efflux. We conclude that in the rat vas deferens high K⁺ promotes tension development by smooth muscle by a dual mechanism: influx of extracellular calcium and release of calcium from internal stores via a IP₃ mechanism.     

Otolith resorption induced by anaerobic stress in the goldfish, Carassius auratus

To examine otolith resorption induced by anaerobic stress, ⁴⁵Ca-prelabelled goldfish, Carassius auratus , were kept in oxygen-deficient ambient water (O₂ < 0.5 .1 1⁻¹, 26° C) for 2 days and otoliths (asterisci) were analysed for ⁴⁵Ca retention. In a second experiment, fish were anaerobically stressed for 2 days, and then received a single intraperitoneal injection of ⁴⁵Ca. They were maintained under stress for one more day and killed to examine ⁴⁵Ca deposition in otoliths. Plasma was analysed for total and radioactive calcium. Otoliths (lapilli) were examined for stress-induced check formation by scanning electron microscopy (SEM).
Stress significantly reduced plasma calcium levels and the rate of calcium retention in otoliths, which was calculated from the ⁴⁵Ca specific activity of the plasma. On the other hand, the rate of calcium deposition in otoliths was the same in the stressed as in the control fish. SEM observation revealed that the applied stress resulted in a check formation in otoliths. These results indicate that a 48-h stress of oxygen deficiency induces calcium resorption in otoliths.     

Mitochondrial calcium uptake in the perfused contracting rat heart and the influence of epinephrine on calcium exchange

Perfused rat hearts were exposed to solutions containing ⁴⁵Ca²⁺ with and without epinephrine. They were subjected to differential centrifugation and the distribution of Ca and ⁴⁵Ca in mitochondria and microsomes was determined. It was found that the mitochondria contain most of the calcium of the intact heart and that the exchange of mitochondrial calcium with extracellular calcium was extremely rapid. This process was accelerated in hearts stimulated by epinephrine.     

Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.     

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC₅₀) and maximal, saturable binding (B_max) were estimated from Scatchard analyses and were found to be 24.2 ± 3.3 pM and 56500 ± 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of 100 nM to a peak level of 600–800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC₅₀ of 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of ⁴⁵Ca from cells loaded to isotopic equilibrium (3 h) with ⁴⁵Ca. The intracellular second messenger, IP₃, also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with ⁴⁵Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 ± 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP₃-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.     

Determination of Vitamin D-dependent calcium absorption by 45Ca gavage in the rat

Precise determination of Vitamin D-dependent intestinal calcium absorption in longitudinal studies is problematic. We have assessed Vitamin D-dependent intestinal calcium absorption by ⁴⁵Ca gavage. Rats were gavaged with a 1 mL solution containing ⁴⁵Ca (CaCl₂, 9.3 MBq/mL) maintained at 37 °C. Total Ca concentration of the gavage fluid was optimised by comparing the absorption curves for fluids made up to 0.025, 2.025, 4.025 and 40.025 mmol/L with ⁴⁰CaCl₂. The effect of varying dietary Ca on fractional Ca absorption was determined in rats fed semi-synthetic diets containing either 0.05%, 0.2%, 0.4% or 1.0% Ca for 50 days. Serum 1,25 dihydroxyvitamin D (1,25D) was determined by radioimmunoassay. Total gavage Ca of 0.025 mmol/L achieved the highest peak fractional absorption and was adopted for all future experiments. Fifty days after allocation to the diets both fractional Ca absorption and 1,25D were highest in rats fed 0.05% Ca and lowest in those fed 1.0% Ca (absorption, P < 0.05 and 1,25D, P < 0.05). There was a strong logarithmic relationship between 1,25D and fractional Ca absorption (R² 0.69, P < 0.001). Weekly repetition of the procedure did not cause a fall in haematocrit over 7 weeks. Radiocalcium (⁴⁵Ca) absorption by gavage provides a simple measure of Vitamin D-dependent Ca absorption for repetitive use in longitudinal studies.     

Effects of rat and chicken calcitonin gene-related peptides (CGRP) upon calcium metabolism in chicks

In vivo effects of intravenously injected chicken(c-) and rat(r-) calcitonin gene related peptide (CGRP) upon plasma total (Ca_t), ionized (Ca_i) calcium, inorganic phosphate (P_i) and clearance of an acutely administered ⁴⁵Ca label have been examined in chicks.

Both peptides were hypercalcaemic in fasted chicks, unlike previously reported hypocalcaemic response in mammals. r-CGRP was hypercalcaemic at doses of both 0.26 and 1.31 nmol/100 g body wt, the lower dose produced a significant elevation of Ca_t one hour after injection into 12-h-fasted chicks, the upper dose had a similar effect at 20 min. Ca_i was also non-significantly elevated by r-CGRP. P_i was slightly increased by r-CGRP at both doses, 20 and 60 min after injection.

c-CGRP produced a dose (0.26–4.17 nmol/100 g body wt) dependent elevation of Ca_t and Ca_i in 22-h-fasted chicks. A greater response was however seen in fed animals. Peak responses were observed 45 min after injection. c-CGRP (1.04 nmol/100 g body wt) caused a significant decline in plasma P_i (P < 0.05) in fasted chicks. P_i was elevated in control fed animals compared with fasted controls. c-CGRP (1.04 nmol/100 g) did not effect plasma P_i in fed chicks.

Whilst both peptides elevated plasma Ca, clearance of an acutely administered ⁴⁵Ca label from plasma was greater in both r-CGRP treated 12-h-fasted chicks and c-CGRP treated 22-h-fasted chicks. In contrast, the rate of ⁴⁵Ca clearance in fed chicks was not affected by c-CGRP treatment.

The differential effects of these peptides upon plasma ⁴⁵Ca clearance and other plasma parameters of Ca metabolism, suggest a complex mode of action of the peptide upon avian Ca homeostasis, possibly involving direct actions upon kidney and bone.     

The effect of diamphenethide on protein synthesis by the liver fluke, Fasciola hepatica

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml⁻¹) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([³H]thymidine, [³H]uridine and [³H] leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [³H]uridine by F. hepatica, decreased the incorporation of [³H] leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (1 × 10⁻³M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [³H] leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin (100 μg ml⁻¹) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [³H]uridine. 5-Fluorouracil (1 × 10⁻⁴ ) did not affect the uptake or incorporation of [³H]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.     

Characteristics of nitric oxide-evoked [H]taurine release from cerebral cortical neurons

Pharmacological characteristics of [³H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. NO generators such as S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) dose-dependently increased [³H]taurine release from neurons. Such stimulatory effects of NO generators were completely abolished by hemoglobin, a NO radical scavenger, indicating that these [³H]taurine releases might be due to NO liberated from SNAP and SNP. Sodium withdrawal from incubation buffer significantly inhibited the SNAP- and SNP-induced [³H]taurine releases, whereas the removal of calcium showed no alterations in the [³H]taurine release evoked by NO generators. β-Alanine and guanidinoethane sulfonate, inhibitors of carrier-mediated taurine transport system, inhibited the SNAP- and SNP-evoked releases of [³H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [³H]taurine release from cerebral cortical neurons is mediated by the reverse process of sodium-dependent carrier-mediated taurine transport system.     

Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [³H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [³H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively, and evoked a 19-fold stimulation in the synthesis of prostaglandin E₂. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [³H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75–80% of the phorbol ester-promoted (total) cellular liberation of [³H]arachidonic acid and production of prostaglandin E₂. In comparison, the release of [³H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA₂ inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe. PMA-induced formation of diacylglycerol or synthesis of PGE₂ was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylgycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.     

Ca-Cd interaction in the prymnesiophyte Cricosphaera elongata

Abstract. ⁴⁵Ca and ¹⁰⁹Cd uptake were followed in Criscosphaera elongata Prymnesiophyceae. In both cases, after a rapid increase for the first 5 min, the incorporation rate slowed during the hour of observation. Verapamil, a blocker of voltage-dependent slow calcium channels, inhibited ⁴⁵Ca uptake except during the first rapid phase when adsorption should predominate. Cadmium also decreased ⁴⁵Ca labelling, suggesting that the two metals are antagonistic. However, verapamil was shown to augment ¹⁰⁹Cd incorporation, contrary to what occurs in animals cells; this effect is detectable in continuous labelling as well as in pulse experiments. The data support the presence of calcium channels in the alga and suggest several processes in Cd accumulation: adsorption on peripheral envelopes and diffusion of uncharged Cd.     

Selective enhancement in striatal [H]nitrendipine binding following chronic treatment with morphine

Two parameters of Ca²⁺ dynamics in brain preparations (⁴⁵Ca-uptake to slices and [³H]nitrendipine binding to membrane fractions) were measured in naive and chronic morphine-administered rats. While morphine did not have any effect on ⁴⁵Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K⁺-stimulated ⁴⁵Ca-uptake to slices. Furthermore, the effect of morphine was antagonized by naloxone. Inhibition of K⁺-stimulated ⁴⁵Ca-uptake to striatal slices by morphine was not observed in preparations obtained from chronic morphine-administered rats (6 mg/kg/b.i.d./7 days). In membrane fractions, [³H]nitrendipine binding increased by 34% in striatum following chronic morphine treatment, whereas no change was observed in the cortex and hippocampus. The results will be discussed in relation to the phenomena underlying chronic morphine administration.     

Binding activities by propiverine and its N-oxide metabolites of L-type calcium channel antagonist receptors in the rat bladder and brain

The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N → O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N → O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N → O) inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the K_i value for P-4(N → O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly K_d values for bladder (+)-[³H]PN 200–110 binding. DPr-P-4(N → O) had little inhibitory effects on the bladder (+)-[³H]PN 200–110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-³H]scopolamine methyl chloride ([³H]NMS) binding in the rat bladder. The ratios of K_i values for (+)-[³H]PN 200–110 to [³H]NMS were markedly smaller for propiverine and P-4(N → O) than oxybutynin and DEOB. Propiverine and P-4(N → O) inhibited specific binding of (+)-[³H]PN 200–110, [³H]diltiazem and [³H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The K_i values of propiverine and P-4(N → O) for [³H]diltiazem were significantly smaller than those for (+)-[³H]PN 200–110 and [³H]verapamil. Further, their K_i values for [³H]verapamil were significantly smaller than those for (+)-[³H]PN 200–110. The K_i values of propiverine for each radioligand in the cerebral cortex were significantly (P < 0.05) smaller than those of P-4(N → O). In conclusion, the present study has shown that propiverine and P-4(N → O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

Activation of phospholipase D by endothelin-1 and other pharmacological agents in rabbit iris sphincter smooth muscle

The stimulation of phospholipase D (PLD) activity by endothelin-1 (ET1) was investigated in rabbit iris sphincter perlabelled with [³H]myristic acid. In the presence of 0.5% ethanol, ET1 caused a time- and dose-dependent increase in the production of [³H]phophatidylethanol ([³H]PEt). Within 30 s the peptide increased PEt formation by 30% and after 5 min increased it by 140%. The ₅₀ value for ET1-stimulated PEt formation was found to be 30 nM. This value is appreciably lower than the ₅₀ we previously obtained for ET1-induced inositol triphosphate production (45 nM), but considerably higher than that for arachidonic acid release (1 nM). PEt formation was significantly stimulated by prostaglandin F₂₀, phorbol 12,13-dibutyrate (PDBu), chloroform, A23187 and A1F₄⁻, but it was not affected by carbachol or the platelet-activating factor. PDBu-stimulated PEt formation was blocked by staurosporine and it was not potentiated by A23187. Staurosporine had no effect on ET1-stimulated PEt formation. Our data indicate that ET1 stimulation of PLD occurs independently of protein kinase C activation, phospholipase C activation and intracellular Ca²⁺ mobilization, and phospholipase A₂ activation. In this tissue the ET1 receptor is probably coupled to the three phospholipases through several G-proteins, and this appears to be species and receptor type specific.     

A differential interaction of putative μ-selective agonists with opiate binding sites in rat brain

The availability of tritium-labelled sufentanil ([³H]SUF) allowed for a further radioligand analysis of opiate binding sites in rat brain. A comparison of the binding characteristics of [³H]SUF and [³H]dihydromorphine ([³H]DHM) revealed a very similar potency in their mutual displacement by unlabelled analogues. Furthermore, a series of putative μ-opiate agonists displayed equal potencies in displacing either [³H]SUF and [³H]DHM, the only striking exception being the highly μ-selective opioid peptide morphiceptin which was 33 times less potent in inhibiting [³H]SUF as compared to [³H]DHM binding. Additional experiments revealed further pronounced differences in [³H]SUF and [³H]DHM binding characteristics: the total amount of binding sites for [³H]SUF was 4 times higher than that for [³H]DHM and the regional distribution within particular brain areas displayed considerable differences. Furthermore, the binding of [³H]SUF was differentially modulated by sodium and GTP as compared to [³H]DHM binding. These data suggest that in rat brain, [³H]SUF interacts both with μ-opiate sites recognizing [³H]DHM and another type of opiate site, which cannot be equated with any of the, as yet, described δ- or κ-binding sites, and rather, represents a subclass of μ-opiate receptor sites. These experiments, thus, support the notion of subclasses (isoreceptors) for different types of opiate receptors.     

Role of antioxidants in the nitric oxide-elicited inhibition of dopamine uptake in cultured mesencephalic neurons. Insights into potential mechanisms of nitric oxide-mediated neurotoxicity

Under aerobic conditions the addition of (C₂N₅)₂N(N[O]NO)⁻ · Na⁺(DEA/NO), S-nitroso-N-macetyl penicillamine and nitric oxide (NO)-saturated buffer, but not S-nitroso- -glutathione, to dopamine solutions resulted in dopamine o-semiquinone formation that was dependent on the formation of a NO/oxygen intermediate. High pressure liquid chromatography (HPLC) electrochemical analysis of dopamine demonstrated that the DEA/NO-induced oxidation of dopamine was abrogated in the presence of the antioxidants, ascorbate and glutathione. NO spontaneously released from DEA/NO decreased [³H]dopamine accumulation in primary cultures of mesencephalic neurons in a dose-dependent fashion. In contrast, [³H]γ-aminobutyric acid uptake by mesencephalic neurons tested under the same conditions was unchanged. When DEA/NO was added to incubation buffer that contained [³H]dopamine and the antioxidant, ascorbate or glutathione, [³H]dopamine uptake was also inhibited. These data excluded that oxidation of extracellular [³H]dopamine by the intermediates of the NO/O₂ reaction could have caused this decrease. Instead, NO may have acted directly on a not yet identified target operative in the regulation of dopamine storage and release. Analysis of the rate constants for the NO reaction with ascorbate, glutathione and dopamine revealed that dopamine quinone formation was delayed by the presence of antioxidants. Since the formation of NO as well as neurotransmitter release are activated during ischemia reperfusion injury, it is possible that prolonged NO exposure could deplete antioxidants and facilitate the oxidation of dopamine and thereby cause neurotoxicity.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Acylation of myelin proteolipid protein in subcellular fractions of rat brainstem

The acylation of myelin proteolipid protein (PLP) and intermediate protein (IP) was investigated in an in vitro system of tissue slices prepared from actively myelinating rat brainstem. The incorporation of [³H]palmitate into the proteins in nine subcellular fractions including myelin and other cellular membranes which are actively involved in the synthesis and intracellular transport of the proteins was measured. More than 80% of [³H]palmitate-labeled proteins were recovered in myelin. The incorporation was highest in the heavy myelin and lowest in the light myelin subfraction. Appreciable acylation was also detected in the myelin-like fraction. On the other hand, the remaining fractions comprising a variety of endo- and ectomembranes, which harbored over 90% of newly synthesized PLP and IP as seen from [³H]leucine labeling showed practically no [³H]palmitate incorporation. The results indicate that the acylation of PLP and IP is a late event in their posttranslational processing and occurs only at their entry into the myelin sheath.