Presynaptic opioid receptors modulating acetylcholine release in the hippocampus of the rabbit

Summary Inhibition of cardiae adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [³H]ouabain binding, whereas 5-nucleotidase activity and -adrenoceptor binding displayed an additonal peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N⁶-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A₁-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[¹²⁵I]iodo-N⁶-hydroxyphenyl-isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A₂-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum.Abbreviations R-PIA (–)N⁶-(R-phenylisopropyl)-adenosine - NECA 5-(N-ethyl-carboxamido)-adenosine - ICYP (–)[¹²⁵I]iodo-cyanopindolol - dATP 2-deoxy-adenosine-5-triphosphate - S-PIA (+)N⁶-(S-phenylisopropyl)-adenosine - HPIA (–)N⁶-(4-hydroxy-phenylisopropyl)-adenosine - CHA N⁶-cyclohexyl-adenosine - Gpp(NH)p guanylyl imidodiphosphate - dAMP 2-deoxy-adenosine-5-monophosphate - HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid - EDTA (ethylenedinitrilo)-tetraacetic acid - [¹²⁵I]HPIA (–)N⁶-(3-[¹²⁵I]-iodo-4-hydroxyphenylisopropyl)-adenosine     

An excitatory input to nucleus raphe magnus from the red nucleus in the cat

In chloralose-anaesthetized cats, with the cerebellum removed, stimulation in the red nucleus excited the majority (60-65%) of neurones in nucleus raphe magnus (NRM), including raphespinal neurones. Evidence was obtained for both monosynaptic and polysynaptic excitation. The projection was confirmed by recording antidromic responses in the red nucleus to stimulation in NRM. It is suggested that the role of NRM in motor control is to inhibit spinal flexion responses to peripheral stimuli so that commands from the red nucleus and other motor control regions may take place without interruption.     

Effect of glucose-6-phosphate and pH on glucose transport in skeletal muscle plasma membrane giant vesicles

The effect of glucose-6-phosphate (G-6-P) and pH on glucose transport was studied in skeletal muscle plasma membrane giant vesicles containing GLUT4 but not GLUT1. Vesicles (average diameter 7.6 μm) were obtained by collagenase treatment of muscle. The vesicles were incubated with 10 mmol T^-1 G-6-P and, after 0.5 and 2 h of incubation, the intravesicular G-6-P concentration was 0.93 ± 0.4 mmol 1^-1 and 1.18 ± 0.5 mmol I“¹ (mean ± SE, n = 4), respectively. In order to increase the intravesicular G-6-P concentration, 0.001% saponin was added during incubation, which increased the 2-h intravesicular G-6-P concentration to 4.57±1.0 mmol 1^-1 (n = 4). Initially, vesicles were used for glucose transport studies after 30 min of incubation with 10 mmol 1^-1 of G-6-P. There was no effect of G-6-P on either the affinity constant (K_m) or maximal velocity (V_max) of the glucose transport. Subsequently, vesicles were incubated for 2 h with 10 mmol T^-1 of G-6-P and 0.001 % saponin. Still no effect of G-6-P on glucose transport could be detected. In contrast, the rate of D-glucose transport was affected, when extravesicular pH was varied from 6.0 to 7.8. The maximum glucose transport rate was found at pH 7.2 and was decreased at both higher and lower pH. It is concluded that G-6-P has no effect on GLUT4 intrinsic activity in rat skeletal muscle plasma membrane. In contrast, GLUT4 intrinsic activity is sensitive to changes in pH.     

Regulation of sarcolemmal Ca2+ pump by endogenous protein phosphatases

Summary The function of several key sarcolemmal proteins is modulated through phosphorylation-dephosphorylation of serine/threonine residues. While the involvement of sarcolemma-associated protein kinases in the phosphorylation of these proteins has been established, the nature of the protein phosphatases controlling these proteins has not been investigated. Rat heart sarcolemma contains two protein phosphatase isozymes, protein phosphatase 1 and 2A, which are distinguished on the basis of their susceptibility of inhibitor 2. Both isozymes elute from a Bio Gel A-0.5 column in association with the highest molecular weight protein fraction. However, some protein phosphatase 1 activity elutes with a smaller molecular weight fraction of about 37000, suggesting that the native enzyme exists as a catalytic subunit in complex with an anchor protein. Inhibition of the protein phosphatases using standard inhibitors leads to a stimulation in both the rate and extent of³²P incorporation into isolated sarcolemma. Also affected by inhibition of protein phosphatase activity is the rate of ATP-dependent calcium uptake, which is stimulated following exposure to either inhibitor 2, a classical protein phosphatase 1 inhibitor, and microcystin, a protein phosphatase 1 and 2A inhibitor. The data suggest that the protein phosphatases regulate the dephosphorylation of sarcolemmal proteins Through this mechanism they serve as important modulators of the sarcolemmal Ca²⁺ pump.     

The sarcolemma in the Large(myd) mouse

In the Large(myd) mouse, dystroglycan is incompletely glycosylated and thus cannot bind its extracellular ligands, causing a muscular dystrophy that is usually lethal in early adulthood. We show that the Large(myd) mutation alters the composition and organization of the sarcolemma of fast-twitch skeletal muscle fibers in young adult mice. Costameres at the sarcolemma of the tibialis anterior muscle of Large(myd) mice contain reduced levels of several membrane cytoskeletal proteins, including dystrophin and beta-spectrin. In the quadriceps, longitudinally oriented costameric structures tend to become thickened and branched. More strikingly, proteins of the dystrophin complex present between costameres in controls are absent from Large(myd) muscles. We propose that the absence of the dystrophin complex from these regions destabilizes the sarcolemma of the Large(myd) mouse and thereby contributes to the severity of its muscular dystrophy. Thus, the positioning of sarcolemmal proteins may have a profound effect on the health of skeletal muscle.     

Fuzzy space and control of Na+, K+-pump rate in heart and skeletal muscle

Since intracellular Na⁺ activity (aⁱ_Na) is one important determinant of Na⁺, K⁺-pump rate as well as excitability and the finely tuned contractility, it is surprising that the relation between aⁱ_Na and pump rate reported by different authors has k_0.5 varying between 10 and 40 mmol L^-1. Other data also point to a variable relation between pump rate and aⁱ_Na. During stimulation of isolated rat soleus muscles at 2 Hz, ouabain-sensitive ⁸⁶Rb uptake was increased in spite of the intracellular Na⁺ remaining unaltered. In isolated cardiomyocytes, a transient Na⁺, K⁺-pump current was observed upon activation by extracellular K⁺ in spite of good control of aⁱ_Na. Na⁺-loaded, isolated, sheep cardiac Purkinje fibres initially hyperpolarized over a period of up to 1 min upon activation of the Na⁺, K⁺ pump with no detectable change of aⁱ_Na. These examples are compatible with the existence of a micro-environment close to the membrane where diffusion is slower than in the rest of the cytosol, so that local aⁱ_Na may fluctuate or gradients may develop as visualized by Wendt-Gallitelli et al. (1993). We conclude that the reported relationships between Na⁺, K⁺-pump rate and aⁱ_Na in intact cells probably underestimate the true affinity of the Na⁺, K⁺ pump for Na⁺ due to a functional diffusion barrier beneath the sarcolemma, and also because of incomplete cell dialysis in whole-cell voltage clamp experiments. The Na⁺, K⁺ pump seems to be preferentially supplied with Na⁺ from the outside through neighbouring channels and transporters.     

Phospholipase A activity of highly enriched preparations of cardiac sarcolemma from hamster and dog.

Cardiac sarcolemma was isolated from hamster and dog and was examined for phospholipase A activity using 1-acyl [2-¹⁴C]-linoleoyl3-glycerophosphorylethanolamine as substrate. Hamster sarcolemmal preparations contained phospholipases A₁ which had optimal activity at pH 6.0 and pH 9.0 in the presence of 5 mm Ca²⁺; EDTA was a potent inhibitor of phospholipase activity at both pHs. The specific activities of the hamster sarcolemmal phospholipases A₁ were increased 5.9-fold (pH 6.0) and 8.4-fold (pH 9.0) over the homogenate, while the increase in specific activity of the sarcolemmal marker enzyme, ouabain-sensitive (Na⁺-K⁺)-Mg⁺-ATPase, was 7.5-fold; thus both the (Na⁺+K⁺)-Mg⁺-ATPase and phospholipase A₁ activities were associated with enriched cardiac sarcolemmal membranes. Canine myocardial sarcolemmal preparations also contained a phospholipase A that had optimal activity at pH 7.0 in the presence of 5 mm Ca²⁺ and the enzyme exhibited apparent specificity for the 2-position. The phospholipase A and (Na⁺+K⁺)-Mg²⁺-ATPase activities were similarly enriched in the canine sarcolemmal preparation. These endogenous, calcium-stimulated phospholipase A activities may be important constituents of the myocardial sarcolemmal membrane; they may modulate the lipid environment of the sarcolemma and may regulate the activity of lipid-dependent enzymes as well as alter membrane permeability.