Ion-Selective Electrodes for Measuring Potassium in Erythrocytes: a Model for Clinical Interpretation of the Results (a Pilot Study).

The aim of the investigation is to study the concentration of potassium in erythrocytes using the proposed method, potassium interconnection with other electrolyte and acid-base parameters of blood plasma, and to create the basis for clinical interpretation of the results. Materials and Methods: Potassium content in erythrocytes was measured using a blood gas analyzer with ion-selective electrodes in parallel with the laboratory procedure. Patients from intensive care units were randomly selected for the study. Results: No correlations of potassium with other plasma parameters have been found, however its buffer dependence on chlorine in plasma has been established. Minimal value of potassium concentration in erythrocytes (for 356 measurements) was 68.2 mmol/L, maximal - 210.2 mmol/L.Following the logic of the acid-base status, a nomogram for clinical interpretation of intracellular potassium homeostasis has been developed. The low values are mainly connected with the deficit of potassium which is impossible to determine in blood plasma (e.g. in severe metabolic alkalosis or diuretic therapy). The elevated concentration of potassium in erythrocytes is caused by eryptosis: released potassium is absorbed by normal erythrocytes (protection from hyperkalaemia). So, the increased concentration of potassium indicates directly the presence of eryptosis triggers, i.e. inflammatory mediators, oxidative stress, and others, for example in sepsis. The results of the study have shown that measurement of potassium concentration in erythrocytes with the help of ion-selective electrodes is an effective method of monitoring its intracellular homeostasis. Potassium in erythrocytes is an independent biological marker which can provide clinically relevant information.

Microtubules are involved in the localization of tau mRNA in primary neuronal cell cultures.

Subcellular localization of neuronal mRNAs contributes to the development of identifiable microdomains. In differentiated neurons, tau mRNA is localized in the cell body and the proximal portion of the axon, and MAP2 mRNA is localized in the cell body and dendrites, whereas tubulin mRNA is restricted to the cell body. To investigate the mechanism(s) leading to segregation of mictrotubule-associated protein mRNA, we examined the role of the cytoskeleton in this process. Detergent extraction of primary neuronal cells in culture followed by in situ hybridization analysis demonstrated that tau mRNA remains bound to cytoskeleton of the treated cells. In addition, biochemical fractionation showed that tau and MAP2 mRNAs are preferentially associated with the fraction of assembled microtubules. In contrast, mRNAs restricted to the neuronal cell body, such as those of tubulin, the 68 kDa neurofilament, and mouse GAPDH, are preferentially found in the supernatant. Using cytoskeletal inhibitors, we demonstrate that tau mRNA is associated with the microtubule system, and not with the actin filaments, thus supporting the hypothesis that the mechanism of mRNA localization is a multistep pathway in which the microtubules play a crucial role.

Subcellular localization of tau mRNA in differentiating neuronal cell culture: implications for neuronal polarity.

A primary neuronal cell culture derived from whole brains of fetal rats was used to analyze the subcellular localization of tau mRNA, employing nonisotopic detection by in situ hybridization. The culture exhibited a developmental differentiation pattern previously described for neuronal cells in vivo; i.e., a transition from immature to mature tau isoforms as well as segregation of tau into the axons. Our results demonstrate that unlike tubulin mRNA, which is confined to cell bodies, or MAP2 mRNA, which extends into dendrites, tau mRNA was observed to enter the proximal portion of the axon. This sorting of tau mRNA might explain how the tau protein could be selectively delivered to the axon and could have important implications for the development of neuronal polarity.

Complete sequence of 3'-untranslated region of Tau from rat central nervous system. Implications for mRNA heterogeneity.

Tau is a family of microtubule associated proteins, heterogeneous in molecular weights, which are expressed specifically in neurons. Tau is encoded by a single gene, while its transcript undergoes a complex and regulated alternative splicing, giving rise to several mRNA species that migrate on Northern blots at approximately 6 and 2 kb. In this report we characterize a full size transcript of tau mRNA from rat brain and demonstrate that it contains 5203 nucleotides (not including exon 2 and 3), which correlates well with the exact size of the transcript as analyzed by Northern blot using RNA standard size markers. The full length of the 3'-untranslated region contains 3848 bp and includes two polyadenylation signals which may yield the two size transcripts in the central nervous system. The first polyadenylation signal is located in the retained intron 13/14 and the second polyadenylation signal is 19 nucleotides before the poly(A) tail. Unspliced intron 13/14 was detected in all RNA preparations tested, including RNA from different ages and different regions of rat brain, RNA from dorsal root ganglia and from undifferentiated and differentiated PC12 cells. In none of the above tissues and cells was a spliced transcript lacking intron 13/14 detected.

Identification of a tau promoter region mediating tissue-specific-regulated expression in PC12 cells.

Tau, a microtubule-associated protein, is encoded by a single gene, the expression of which is neuron-specific and developmentally regulated. When PC12 cells are exposed to nerve growth factor (NGF), they differentiate to sympathetic-like neurons. This differentiation process is accompanied by an elevation of tau proteins and mRNA. Here, we describe, for the first time, the isolation and characterization of a tau promoter region. We show that the promoter of tau is G + C-rich, lacks a genuine TATA box and thus promotes multiple initiation sites of RNA transcription. Our results demonstrate that a region of approximately 335 base-pairs residing immediately upstream of tau exon -1 are able to direct positive control of neuron-specific activity of the luciferase reporter gene. The isolation of tau promoter will facilitate facilitate further studies of the regulation of tau expression during development and aging of neuronal cells.

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling.

Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N-type voltage-dependent Ca2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricyclic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the Gi/G(o) GTP-binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and delta 9-tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.

kappa-Opioid receptor-transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments.

The opioid receptors mu, delta and kappa have recently been cloned. Here we show that kappa-agonists inhibit adenylyl cyclase activity in Chinese hamster ovary cells stably transfected with rat kappa-opioid receptor cDNA. Chronic exposure of the cells to kappa-agonists did not lead to significant desensitization of the capacity of the agonists to inhibit adenylyl cyclase. On the other hand, withdrawal of the agonist following the chronic treatment led to the phenomenon of supersensitivity ('overshoot') of adenylyl cyclase activity. Both the inhibition of adenylyl cyclase activity by the acute opioid treatment and the chronic agonist-induced supersensitivity are pertussis toxin sensitive, demonstrating involvement of Gi/Go proteins in both processes.

Hyperinsulinaemia increases blood pressure in genetically predisposed spontaneously hypertensive rats but not in normotensive Wistar-Kyoto rats.

BACKGROUND: There is controversy in the literature concerning the effect of short-term insulin administration on blood pressure in different experimental situations, because in some experiments this association is clear, whereas in others it is nonexistent. OBJECTIVE: To investigate whether there is a difference in the effect of exogenous insulin administration on the blood pressure of normotensive Wistar-Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHR). METHODS: Hyperinsulinaemia was induced in normotensive WKY rats and in hypertensive SHR by the administration of long-acting insulin (insulin retard 0.4 U/kg body weight per day in one group and 0.8 U/kg body weight per day in another group) once a day, intraperitoneally, for 3 weeks. All of the rats drank a 10% sucrose solution, to prevent hypoglycaemia in those receiving insulin. RESULTS. Baseline serum levels were significantly higher in the SHR groups than in the WKY rat groups. At the end of the experiment, after 3 weeks' insulin therapy, systolic blood pressure measured by the tail-cuff method showed a significant increase in the SHR, but not in the WKY rats, possibly because of the genetic predisposition of the SHR to increase their blood pressure. The increase was similar in the SHR given 0.4 U/kg body weight per day insulin retard to that in those given 0.8 U/kg per day. CONCLUSIONS: Exogenous insulin increased systolic blood pressure in the SHR but not in the WKY rats. The rise was similar in rats receiving either 0.4 or 0.8 U/kg body weight per day insulin retard.

Cannabinol derivatives: binding to cannabinoid receptors and inhibition of adenylylcyclase.

Several derivatives of cannabinol and the 1,1-dimethylheptyl homolog (DMH) of cannabinol were prepared and assayed for binding to the brain and the peripheral cannabinoid receptors (CB1 and CB2), as well as for activation of CB1- and CB2-mediated inhibition of adenylylcyclase. The DMH derivatives were much more potent than the pentyl (i.e., cannabinol) derivatives. 11-Hydroxycannabinol (4a) was found to bind potently to both CB1 and CB2 (Ki values of 38.0 +/- 7.2 and 26.6 +/- 5.5 nM, respectively) and to inhibit CB1-mediated adenylylcyclase with an EC50 of 58.1 +/- 6.2 nM but to cause only 20% inhibition of CB2-mediated adenylylcyclase at 10 microM. It behaves as a specific, though not potent, CB2 antagonist. 11-Hydroxycannabinol-DMH (4b) is a very potent agonist for both CB1 and CB2 (Ki values of 100 +/- 50 and 200 +/- 40 pM; EC50 of adenylylcyclase inhibition 56.2 +/- 4.2 and 207.5 +/- 27.8 pM, respectively).

Increased expression of synapsin I mRNA in defined areas of the rat central nervous system following chronic morphine treatment.

Chronic opiate administration leads to a selective regulation of several cellular proteins and mRNAs. This phenomenon has been viewed as a compensatory mechanism to the opiate signaling leading to the development of opiate addiction. In this study, in situ hybridization histochemistry experiments were employed to investigate the effect of chronic morphine treatment on synapsin I gene expression. We show here for the first time that prolonged morphine exposure causes a selective increase in the mRNA levels of synapsin I in several brain regions which are considered to be important for opiate action. Quantitative analysis of the signals, obtained by hybridization of digoxigenin-labeled antisense RNA probe, revealed a 5.8- and 7-fold increase of synapsin I mRNA levels in the locus coeruleus and the amygdala of morphine-treated rats, respectively, as compared with control untreated rats. Increased expression of synapsin I mRNA was also observed in the spinal cord of morphine-treated rats (by 3.8-fold). Since opiates were shown to attenuate neurotransmitter release and reduce synapsin I phosphorylation, it is suggested that the increase in synapsin I levels would lead to the requirement of higher amounts of opiate agonists to obtain the opiate physiological effects. These results suggest that the increases in mRNA levels of synapsin I in these specific areas can be part of the molecular mechanism(s) underlying opiate tolerance and withdrawal.

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.

The effects of angiotensin II, endothelin-1, and protein kinase C inhibitor on DNA synthesis and intracellular calcium mobilization in vascular smooth muscle cells from young normotensive and spontaneously hypertensive rats.

Angiotensin II (Ang-II) and endothelin 1 (ET-1) are important peptides that induce a prolonged vasoconstriction and enhance proliferation of vascular smooth muscle cells (VSMC). These substances may have an important role in the development of hypertension and atherosclerosis. Our objectives were to determine whether there are inborn differences in the proliferation patterns of VSMC obtained from spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY) by studying the effects of Ang-II and ET-1 on VSMC from those strains before the onset of hypertension, and to evaluate the roles of protein kinase C (PKC) and intracellular Ca2+ in the mechanism of action of ET-1 and Ang-II. VSMC from aortas of young (1- to 2-week-old) SHR and WKY rats were grown as primary cultures in plates for 48 h. The cells were incubated with Ang-II (0.1 to 1000 nmol/L) or ET-1 (0.1 to 100 nmol/L). VSMC were also incubated in the presence of various concentrations of a PKC inhibitor, chelerythrine (0.1-10 nmol/L). Thymidine incorporation into DNA was measured as an indicator of DNA synthesis. Intracellular free Ca2+ was determined by using FURA-2AM. ET-1 and Ang-II caused a marked dose-dependent enhancement of thymidine incorporation into DNA. The responses of VSMC from WKY and SHR to Ang-II and ET-1 were similar. In both strains, chelerythrine caused a dose-dependent suppression in the activity of ET-1 and Ang-II. However, VSMC from SHR incubated in the presence of ET-1 were more susceptible to the inhibitory effect of chelerythrine. Both Ang-II and ET-1 induced an increase of intracellular free Ca2+. ET-1 induced a larger increase than Ang-II (190% and 100% greater than baseline free Ca2+ levels, respectively), in spite of a lower concentration of ET-1 (ET-1 = 30 nmol/L; Ang-II = 100 nmol/L). Ang-II and ET-1 exerted a similar mitogenic effect on primary cultures of VSMC obtained from young SHR before the development of hypertension, compared with WKY. The mitogenic activity of Ang-II and ET-1 was accompanied by an increase of intracellular free Ca2+. The effect of ET-1 upon intracellular Ca2+ was stronger than that of Ang-II. VSMC cultures of SHR stimulated with ET-1 were more susceptible to PKC inhibition than those of WKY. The similarity of the effects of Ang- II and ET-1 on SHR and WKY does not exclude their role in the pathogenesis of hypertension and atherosclerosis, and further studies should be carried out to determine their role.

Insulin induces medial hypertrophy of myocardial arterioles in rats.

To investigate the effect of hyperinsulinemia on arteriolar hypertrophy, myocardial hypertrophy, and blood pressure, we administered insulin intraperitoneally to SHR and WKY rats for 3 consecutive weeks. To prevent hypoglycemia, the drinking water contained 10% sugar, and to accentuate the blood pressure, their chow contained 8% table salt. Blood pressure was measured by the tail-cuff method. Heart weights were factored with body weights. Arterioles of approximately 100 microns in diameter were examined at the end of the experiment and the vascular wall thickness was factored with the lumen diameter. At the end of 3 weeks, blood pressure rose in the SHR but not in the WKY rats. The heart weights in the WKY normotensive rats did not increase, whereas in the SHR they did. Furthermore, there was a significant rise in vessel wall thickness in the rats that received insulin, whether there was a rise in blood pressure or not and whether they had an increase in heart weight or not. There was a similar rise in blood glucose in all the groups, with slightly more accentuated rise in the SHR that received insulin. Nevertheless the increase in vascular wall thickness occurred only in the groups which received insulin. This seems to preclude the importance of hyperglycemia per se as the causative agent for the increase in vascular wall thickness in this study. The increase was in the form of medial hypertrophy without any sign of atherosclerosis. It seems, therefore, that hyperinsulinemia is associated with hypertrophy of the media of arterioles regardless of the increase in heart weight or the rise in blood pressure.

Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells.

Catecholamine secretion induced by various secretagogues in cultured bovine chromaffin cells has been correlated with Ca2+ influx and intracellular Ca2+ concentrations. Nicotine and high K+ caused prompt secretion of catecholamines from cells. Coincidently, both secretagogues evoked 45[Ca2+] influx with a parallel increase in free intracellular Ca2+ concentration, as determined by Quin 2 fluorescence. However, the rate of return of Ca2+ level to baseline after nicotine stimulation was more rapid than after K+ stimulation. In comparison, stimulation with veratridine produced a slow and prolonged Ca2+ influx accompanied by lower levels of intracellular Ca2+ than those observed after nicotine or K+ stimulation. Yet, during 15 min of stimulation, veratridine induced a substantial catecholamine release, which was larger than that obtained after nicotine or K+ stimulations. The Ca2+ ionophore A23187 (1 microM) induced a pronounced increase in intracellular Ca2+ levels, but did not evoke any significant catecholamine release. Finally, addition of the Ca2+ channel blocker verapamil following stimulation, at a time when intracellular Ca2+ concentration was at its peak level, did not affect the rate of decline in intracellular free Ca2+ concentration but promptly blocked Ca2+ uptake and catecholamine secretion. These findings suggest that the rate of Ca2+ influx, rather than the absolute level of intracellular Ca2+ concentration, determines the rate and extent of catecholamine release.

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.

Characterization and down-regulation of opiate receptors in aggregating fetal rat brain cells.

Aggregating brain cells prepared from embryonic rats bind radioactive opiates in a stereospecific manner. The drug selectivity, receptor content during culturing and down-regulation of these apparent opiate receptors were studied in aggregates prepared from the embryonic hindbrain or forebrain. The receptor content in hindbrain but not forebrain aggregates was increased up to 3-fold after 21 days in culture. Differences between the receptors of the two types of aggregates were also observed in the affinity of opiate alkaloids and D-Ala2,D-Leu5-enkephalin (DADL). The potent opiate alkaloid etorphine induced down-regulation of opiate receptors in aggregates prepared from either brain region whereas DADL was a potent down-regulator in the forebrain but not in the hindbrain aggregates and morphine had no effect in both tissues. The implications of these results concerning the control of various types of opiate receptors in the whole animal are discussed.

Modulation of thymidine incorporation by kappa-opioid ligands in rat spinal cord-dorsal root ganglion co-cultures.

beta-Endorphin, met-enkephalin and several mu-selective opioid agonists were shown to decrease thymidine incorporation into DNA in various neural cell cultures. We now report that the kappa-selective opioid agonists U50488, U69593 and MR2034 modulate [3H]thymidine incorporation into DNA in rat spinal cord-dorsal root ganglion co-cultures. U50488 at 10 microM increased by 60% thymidine incorporation in 6-day-old cultures. The thymidine incorporation induced by U50488 was blocked by the kappa-selective antagonist nor-binaltorphimine, as well as by pertussis toxin and LiCl. U50488 treatment stimulated phosphatidylinositol turnover by three-fold compared with untreated controls. These findings suggest that kappa-opioid agonists modulate DNA synthesis in spinal cord-dorsal root ganglion co-cultures through a mechanism which involves pertussis toxin-sensitive GTP-binding proteins, as well as activation of phosphatidylinositol turnover.

Muscarinic control of amyloid precursor protein secretion in rat cerebral cortex and cerebellum.

It was previously shown by us and by others that activation of muscarinic acetylcholine receptors evoke amyloid precursor protein (APP) secretion in various cell lines. Here we examined if such muscarinic control of APP secretion occurs also in normal brain tissues. We found that the secretion of APP from rat cerebrocortical slices (rich in M1 receptors) was significantly increased by K+ depolarization, the non-selective agonist, carbachol (CCh), and the M1-selective agonist, AF102B. CCh also increased APP secretion from cerebellar slices (rich in M2 receptors) while AF102B had no significant effect in this brain region. Despite of its stimulatory effect on APP release in the cerebellum, CCh had no effect on phosphoinositide (PI) metabolism in this brain region. In the cerebral cortex PI metabolism was significantly increased by CCh but only partially increased by AF102B. These results suggest that APP secretion in the brain is mediated via muscarinic receptors. In the cerebral cortex APP secretion seems to be regulated via M1 receptors. Our results also suggest that PI metabolism is not a pronounced step in mediating APP processing.

Opioid receptor density changes in Alzheimer amygdala and putamen.

Since opioids can influence the release of acetylcholine, substance P and a number of other neurotransmitters that have been implicated in the pathogenesis of Alzheimer's disease (AD), it is of interest to assess opioid receptor levels in AD. We have examined mu, delta and kappa opioid receptor binding parameters, binding sensitivity to a GTP analog and distribution in amygdala, frontal cortex and putamen of AD brain. Control brains were matched according to age, sex, post-mortem interval and storage time. Kd values and GTP analog binding sensitivity did not differ in AD and control brains. Bmax values for mu ([3H]DAMGE) sites also appeared unaffected by in vitro binding assays. In contrast, kappa ([3H]U69593) and delta ([3H]DSLET) opioid receptor levels, were significantly changed. In AD amygdala kappa Bmax values increased from control levels of 123 +/- 12 to 168 +/- 13 fmol/mg protein, whereas densities of kappa and delta sites were decreased from 94 +/- 8 to 48 +/- 8 and 102 +/- 3.6 to 69 +/- 8.5 fmol/mg protein, respectively, in putamen. Autoradiography revealed corresponding differences in the distribution of kappa opioid receptors. The findings indicate that the kappa binding site, which is quantitatively the major opioid receptor class in human brain, undergoes marked changes in AD amygdala and putamen.

Expression of the three opioid receptor subtypes mu, delta and kappa in guinea pig and rat brain cell cultures and in vivo.

Expression of the three opioid receptor subtypes mu, delta and kappa in aggregating cell cultures prepared from embryonic guinea pig or rat brains was compared with the in vivo expression of the receptors in the brain of developing and adult animals of the same species. At the day of culturing, one third of the receptors in the brain of guinea pig embryos were of the kappa type. In culture, however, the aggregating brain cells acquired within 14 days a high percentage (75%) of kappa receptors. As only 28% of the receptors in the adult guinea pig brain are of this subtype, an attempt was made to further analyse the specificity of this developmental process. In guinea pig, the 2-fold increase in kappa receptors in culture was accompanied with a decline in both the percentage and the density (per protein) of mu and delta subtypes. In contrast, a marked increase in delta receptors was observed in rat whole brain, forebrain or hindbrain cultures. Thus, the developmental pattern of the three receptor subtypes in rat brain cultures, but not in guinea pig, was similar to that in vivo. These and additional experiments suggest that at the developmental stage taken to prepare the cultures, neurons expressing opioid receptors were already programmed in the rat but not in guinea pig brain.