Biochemical similarity of rat pituitary and CNS TRH receptors

Chemical properties of receptor binding sites for thyrotropin-releasing hormone (TRH) in rat pituitary, retina, amygdala and hypothalamus were compared by examining the influence of sulfhydryl reagents on specific binding of [3H](3-Me-His2)-TRH ([3H]MeTRH). Dithiothreitol-induced reduction of disulfide bonds, alkylation of thiol residues by N-ethylmaleimide and their oxidation by 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), all produced marked reduction of [3H]MeTRH binding, which could be prevented in part by preincubation with exogenous TRH. In all tissues, concentration-dependent loss of binding activity was observed following exposure to micromolar heavy metals and mono- and divalent cations, with apparent additive effects between cations and DTT and NEM. Most changes appeared to reflect only a decrease in receptor density (Bmax). The similar sensitivity of all tissues to these compounds complements existing evidence for a close resemblance of TRH receptors in the CNS and pituitary.     

Calmodulin infused intracerebroventricularly enhances food intake in the cat

In the fasted cat, calmodulin (CaM) infused into the cerebral ventricle produces an increase in the normal intake of food in a dose-dependent manner. The enhancement of feeding by CaM seems to be functionally specific since the response was: (1) abolished by the simultaneous intraventricular infusion of calcineurin, a specific CaM antagonist; (2) not mimicked by another calcium binding protein, troponin C; and (3) independent of the CaM's lack of effect on body temperature and water intake. This finding opens up the dual possibility that this Ca2+ binding protein may affect receptors other than intracellularly and that CaM is involved in specific functions controlled by the brain.     

Preliminary characterization of synaptic vesicle/calmodulin interaction

The association of calmodulin with brain synaptic vesicle proteins was analyzed. Scatchard analysis of [¹²⁵I]calmodulin binding to brain synaptic vesicles revealed one high-affinity, low-binding-capacity,K_d = 1.0 (± 0.15)nM,B_max = 4.1 (± 0.6)pmol/mg, and one low-affinity high-binding-capacity site,K_d = 177. (± 12.0)nM andB_{max= 202 (± 15.0)pmol/mg}. Triton X-100 solubilization of synaptic vesicle proteins and subsequent elution on a Sepharose-4B-CNBr-calmodulin affinity column demonstrated that two protein doublets of approximate M_r^S 55 K and 30 K were the major synaptic vesicle calmodulin binding proteins. In addition there were two minor calmodulin binding singlet polypeptides with M_r^S 62 K and 40 K. Calmodulin stimulated endogenous synaptic vesicle protein kinase, Ca²⁺,Mg⁺-ATPase and Ca²⁺ uptake activities. Phosphorylation assays coupled with immunological studies using affinity-purified antibodies suggested that the synaptic vesicle Ca²⁺/calmodulin-dependent protein kinase migrated in the 30 K M_r region.     

Modulation of striatal cyclic nucleotide phosphodieterase by calmodulin: regulation by opiate and dopamine receptor activation.

Incubation of striatal slices with morphine (10^?6 M) increased the cytosolic content of calmodulin. The double reciprocal plot of cAMP-phosphodiesterase (PDE) activity, vs cAMP concentrations which appears to be biphasic with an apparent low and high K_m-form in control slices was changed to a monophasic one with a low K_m-form. The changes in the apparent K_m for cAMP elicited by morphine (5 × 10^?7 M) could be blocked by haloperidol (10^?7 M) and naltrexone (10^?7 M). In slices prepared from deafferented caudate nuclei, morphine (5 × 10^?7 M) did not cause a change in the biphasic double reciprocal plot, but incubation of such slices with dopamine (2 × 10^?7 M) still caused the appearance of a low K_m-form of phosphodiesterase. Gel filtration (G-150) of soluble extracts indicated that a significantly greater proportion of the calmodulin was found in the fraction containing PDE activity in the morphine-treated slices than in control slices. Similar changes in PDE activity and calmodulin did not occur in cerebellar slices. These findings suggest that opiate receptors may be located on dopaminergic neurons, and that opiates may influence the regulation of PDE by calmodulin via post-synaptic dopamine receptors. Changes in PDE mediated by the increased availability of calmodulin can be used as an index of stimulation of dopamine receptors and may be related to the development of subsensitive receptor responses.     

Platelet calcium-dependent proteins: Identification and localization of the calcium-dependent regulator,calmodulin, in platelets

The calcium-dependent regulator protein, calmodulin, is a 17,000 molecular weight polypeptide which binds calcium and has been shown to confer calcium sensitivity on contractile and other proteins. In the present study, we have examined the presence and subcellular distribution of this protein in preparations of human platelets. Calmodulin was quantified using a two-stage phosphodiesterase assay. Whole platelets contained 1.33 ± 0.06 units calmodulin per 10⁶ platelets or 26.5 ± 3.4 fg calmodulin per platelet. The distribution of calmodulin in the platelet was predominantly soluble with over 80 percent of calmodulin activity in the soluble fraction of the cell. There was no apparent difference in the distribution of calmodulin between soluble and particulate compartments in recalcified platelet homogenates compared to homogenates in EDTA. Indirect immunofluorescent studies with monospecific antisera to dinitrophenylated calmodulin showed intense staining of platelets in a diffuse pattern. The identification of calmodulin in platelets raises the possibility that this protein may participate in calcium-dependent reactions important in platelet aggregation and release.     

Sickle red cell calcium metabolism: studies on Ca2+-Mg2+ATPase and Ca-binding properties of sickle red cell membranes

Sickle (Hb SS) red cells, preloaded with 45Ca by reversal of hemolysis, exhibit an incomplete 45Ca extrusion, retaining approximately four times more 45Ca than normal cells. Studies indicated that neither the reduction in Hb SS cell Ca2+-Mg2+ ATPase activity (84% of normal) nor the activation of Ca2+-Mg2+ ATPase by calmodulin was sufficiently different from normal cells to attribute a major role to the calcium pump in 45Ca retention. These results suggested that 45Ca retention may reflect an alteration in the calcium-binding properties of Hb SS cell membranes. Low-affinity calcium-binding (freely dissociable) was similar in normal and Hb SS cell membranes. However, the total calcium bound with high-affinity (tightly bound) was four-to-five times greater in Hb SS cell membranes than in normal membranes. These results are compatible with the hypothesis that Hb SS cell 45Ca retention reflects an exchange of a fraction of the total 45Ca with a tightly bound calcium pool, larger in Hb SS cell membranes than in normal membranes. A comparable degree of red cell 45Ca retention, which did not correlate with the reticulocyte population, was observed in other chronic anemic states. These findings suggest that the increased high-affinity calcium binding by the membrane may be a consequence of cellular changes induced by the anemic condition.     

Calmodulin,zinc, and calcium in cellular and membrane regulation: An interpretive review

A considerable amount of work in recent years has indicated that the intracellular effects of calcium are mediated primarily by calcium-binding proteins. One of the most important of these proteins is a 17,000-molecular weight protein–calmodulin. Calmodulin, when activated by calcium, has the potential to activate many enzymes and to stimulate many intracellular events. While calcium is required for many types of cellular activations, zinc tends to be a cellular inhibitor. It appears that zinc is antagonistic to calcium, tending to inhibit the same cellular reactions triggered by an influx of intra-cellular calcium. Recent investigations have led us to hypothesize that the zinc-calcium antagonism mentioned above may be the result of inhibitory effects of zinc on calmodulin-stimulated functions. This inhibition may operate through binding of zinc to the target proteins of calmodulin or through binding of zinc to calmodulin after the calmodulin is complexed to protein. This area will be reviewed in the first part of this paper. In the erythrocyte, excessive intracellular calcium causes hemoglobin retention by erythrocyte membranes and causes erythrocyte shrinkage. Both of these events may occur in sickle erythrocytes because of sickling-induced calcium accumulation. Recent studies suggest that excessive calcium-activated calmodulin may be responsible for both hemoglobin retention by the membrane and for erythrocyte shrinkage. On the other hand, trifluoperazine — a phenothiazine drug that inhibits the action of calmodulin — causes erythrocyte swelling (membrane expansion). These observations have led us to hypothesize that calmodulin is intimately involved with regulation of the erythrocyte membrane expansion/shrinkage state. The possible role of excessive calcium-activated calmodulin in inducing abnormalities in the sickle membrane may lead to a more rational therapy aimed at the membrane lesion in this disease. This therapy might involve a combination of inhibitory agents such as zinc and a phenothiazine. In the last part of the review, we present a model for the regulation of membrane shrinkage by calmodulin in equilibrium with membrane expansion by spectrin-actin phosphorylation. Whether or not this model is right in its basic elements, we hope it will provoke some additional thinking and experimental work. In fact, this entire review should be viewed as interpretive. That is, observations from diverse sources have been put together in an attempt to make sense out of them and, in particular, to provoke thinking and stimulate experimental work in these areas.     

阿片类药物对NG108-15细胞Ca2+/钙调蛋白依赖的蛋白激酶II信息通路的作用

目的　观察阿片类依赖时Ca²⁺ 钙调蛋白依赖的蛋白激酶II信息通路的变化。方法　以NG108-15细胞作为体外的细胞模型,分别用竞争性蛋白结合法及放射免疫法、PDE法、γ-³² P参入法测定cAMP水平、钙调蛋白(CaM)活性和钙调蛋白依赖的蛋白激酶II(CaMKII)活性。结果　DPDPE作用NG108-15细胞48h可使细胞浆和细胞核CaM和CaMKII活性升高,该变化可被CaM特异性拮抗剂W-7所抑制;CaMKII特异性抑制剂KN-62可抑制CaMKII活性的增高,而对CaM活性无明显影响。DPDPE作用NG108-15细胞48h后,加入纳洛酮,CaM活性、CaMKII活性进一步增高。结论　Ca²⁺ CaMKII信息通路参与了阿片依赖的机制。     

阿片类药物对NG108-15细胞Ca~(2 )/钙调蛋白依赖的蛋白激酶II信息通路的作用

目的　观察阿片类依赖时Ca2 钙调蛋白依赖的蛋白激酶II信息通路的变化。方法　以NG10 8 15细胞作为体外的细胞模型 ,分别用竞争性蛋白结合法及放射免疫法、PDE法、γ 32 P参入法测定cAMP水平、钙调蛋白(CaM)活性和钙调蛋白依赖的蛋白激酶II(CaMKII)活性。结果　DPDPE作用NG10 8 15细胞 48h可使细胞浆和细胞核CaM和CaMKII活性升高 ,该变化可被CaM特异性拮抗剂W 7所抑制 ;CaMKII特异性抑制剂KN 6 2可抑制CaMKII活性的增高 ,而对CaM活性无明显影响。DPDPE作用NG10 8 15细胞 48h后 ,加入纳洛酮 ,CaM活性、CaMKII活性进一步增高。结论　Ca2 CaMKII信息通路参与了阿片依赖的机制。     

钙调素拮抗剂E6对大鼠神经细胞内钙的影响

目的　观察钙调素拮抗剂E6对神经细胞静息[Ca2 +]i、KCl (5 0mmol·L-1)和谷氨酸 (glutamicacid ,Glu ,0 1mmol·L-1)引起的神经细胞 [Ca2 +]i 升高的影响。方法　用Ca2 +敏感荧光指示剂Fura 2 /AM负载大鼠脑细胞 ,测定神经细胞内游离Ca2 +浓度 ([Ca2 +]i)。结果　E6可升高神经细胞静息 [Ca2 +]i,EC50 为 6 6 8μmol·L-1;无外Ca2 +条件下 ,也升高 [Ca2 +]i,EC50 为 1 30 μmol·L-1,说明E6主要是通过促进细胞内贮存Ca2 +释放引起内Ca2 +升高。E6抑制KCl升高细胞 [Ca2 +]i 的IC50 为 6 0 μmol·L-1;抑制Glu激发的细胞 [Ca2 +]i 升高的IC50 为 0 15 μmol·L-1。结论　E6可能是通过与细胞内钙调素 (Calmodulin ,CaM)结合后 ,影响兴奋性氨基酸受体的开放和电压依赖性钙通道的活性状态 ,表现出对由去极化或受体激动剂引起的内Ca2 +升高的抑制作用