Use of alpha 2 adrenoreceptor agonists and antagonists in the functional assessment of the sympathetic nervous system.

We studied the effects of clonidine, an alpha 2-adrenoreceptor agonist, and yohimbine, an alpha 2-adrenoreceptor antagonist, on blood pressure, heart rate, and plasma catecholamines in 12 patients with autonomic dysfunction. Clonidine (0.1 mg, orally) lowered blood pressure 18 +/- 3 torr in six subjects and raised it 5 +/- 1 torr in six. The change in blood pressure in response to this dose of clonidine was inversely proportional to the supine level of norepinephrine (P less than 0.05). Yohimbine (4-64 micrograms/kg) raised plasma norepinephrine and blood pressure in six patients with degenerative autonomic dysfunction. Yohimbine also attenuated by 50% (P less than 0.05) the hypotensive response to head-up tilt of patients with degenerative autonomic dysfunction. Clonidine depends upon postjunctional hypersensitivity and the degree of autonomic insufficiency to elicit its pressor response. In contrast, the pressor response to yohimbine is related to the capacity of the sympathetic nervous system to be activated and release norepinephrine. If plasma norepinephrine levels following yohimbine administration are monitored, the biochemical and hemodynamic response to the drug may provide a sensitive indication of the capacity of the sympathetic nervous system to be activated in patients with autonomic dysfunction.

Reprogrammable recognition codes in bicoid homeodomain-DNA interaction.

We describe experiments to determine how the homeodomain of the Drosophila morphogenetic protein Bicoid recognizes different types of DNA sequences found in natural enhancers. Our chemical footprint analyses reveal that the Bicoid homeodomain makes both shared and distinct contacts with a consensus site A1 (TAATCC) and a nonconsensus site X1 (TAAGCT). In particular, the guanine of X1 at position 4 (TAAGCT) is protected by Bicoid homeodomain. We provide further evidence suggesting that the unique arginine at position 54 (Arg 54) of the Bicoid homeodomain enables the protein to recognize X1 by specifically interacting with this position 4 guanine. We also describe experiments to analyze the contribution of artificially introduced Arg 54 to DNA recognition by other Bicoid-related homeodomains, including that from the human disease protein Pitx2. Our experiments demonstrate that the role of Arg 54 varies depending on the exact homeodomain framework and DNA sequences. Together, our results suggest that Bicoid and its related homeodomains utilize distinct recognition codes to interact with different DNA sequences, underscoring the need to study DNA recognition by Bicoid-class homeodomains in an individualized manner.

Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

Cooling-evoked hemodynamic perturbations facilitate sympathetic activity with subsequent myogenic vascular oscillations via alpha2-adrenergic receptors.

This study extends our previous work by examining the effects of alpha2-adrenoceptors under cold stimulation involving the increase of myogenic vascular oscillations as increases of very-low-frequency and low-frequency of the blood pressure variability. Forty-eight adult male Sprague-Dawley rats were randomly divided into four groups: vehicle; yohimbine; hexamethonium+yohimbine; guanethidine+yohimbine. Systolic blood pressure, heart rate, power spectral analysis of spontaneous blood pressure and heart rate variability and spectral coherence at very-low-frequency (0.02 to 0.2 Hz), low-frequency (0.2 to 0.6 Hz), and high-frequency (0.6 to 3.0 Hz) regions were monitored using telemetry. Key findings are as follows: 1) Cooling-induced pressor response was attenuated by yohimbine and further attenuated by hexamethonium+yohimbine and guanethidine+yohimbine, 2) Cooling-induced tachycardia response of yohimbine was attenuated by hexamethonium+yohimbine and guanethidine+yohimbine, 3) Different patterns of power spectrum reaction and coherence value compared hexamethonium+yohimbine and guanethidine+yohimbine to yohimbine alone under cold stimulation. The results suggest that sympathetic activation of the postsynaptic alpha2-adrenoceptors causes vasoconstriction and heightening myogenic vascular oscillations, in turn, may increase blood flow to prevent tissue damage under stressful cooling challenge.

DNA-XPA interactions: a (31)P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98-F219) of the nucleotide excision repair protein XPA.

Recent NMR-based, chemical shift mapping experiments with the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) suggest that a basic cleft located in the loop-rich subdomain plays a role in DNA-binding. Here, XPA-DNA interactions are further characterized by NMR spectroscopy from the vantage point of the DNA using a single-stranded DNA nonamer, dCCAATAACC (d9). Up to 2.5 molar equivalents of XPA-MBD was titrated into a solution of d9. A subset of (31)P resonances of d9 were observed to broaden and/or shift providing direct evidence that XPA-MBD binds d9 by a mechanism that perturbs the phosphodiester backbone of d9. The interior five residues of d9 broadened and/or shifted before (31)P resonances of phosphate groups at the termini, suggesting that when d9 is bound to XPA-MBD the internal residues assume a correlation time that is characteristic of the molecular weight of the complex while the residues at the termini undergo a fraying motion away from the surface of the protein on a timescale such that the line widths are more characteristic of the molecular weight of ssDNA. A molecular model of the XPA-MBD complex with d9 was calculated based on the (15)N (XPA-MBD) and (31)P (d9) chemical shift mapping studies and on the assumption that electrostatic interactions drive the complex formation. The model shows that a nine residue DNA oligomer fully covers the DNA-binding surface of XPA and that there may be an energetic advantage to binding DNA in the 3'-->5' direction rather than in the 5'-->3' direction (relative to XPA-MBD alpha-helix-3).

Increased vascular beta2-adrenoceptor responsiveness in autonomic dysfunction.

Responsiveness to the vasopressor, vasodepressor and chronotropic effects of several sympathomimetic amines was assessed in 12 patients with severe autonomic dysfunction and in 8 age-matched control subjects. The patients with autonomic dysfunction showed a profound increase in responsiveness to both isoproterenol and phenylephrine as compared with control subjects. The mean bolus dose of isoproterenol required to increase heart rate by 25 beats/min was 0.9 + 0.2 microgram in the patients and 5.4 + 2.1 micrograms in the control subjects. The dose of isoproterenol required to reduce mean blood pressure by 25 mm Hg was 0.3 + 0.2 and 5.2 + 1.8 micrograms, respectively. Thus, although there is a 6-fold increase in responsiveness to the chronotropic effect of isoproterenol in autonomic dysfunction, the responsiveness to the drug's depressor effect is increased 17-fold. This enhanced depressor sensitivity is quite marked, even with oral beta-adrenoceptor agonists. Beta-adrenoceptor agonists must be used with caution in conditions associated with autonomic dysfunction if dangerous hypotension is to be avoided.

Linking molecular and bedside research: designing a clinical research infrastructure.

For 40 years the General Clinical Research Centers (GCRCs) have provided the infrastructure in Academic Medical Centers (AMCs) in the United States, which enabled and facilitated the extraordinary achievements of twentieth century patient-oriented research. The GCRC program in the United States represents a central theme linking molecular and clinical research directed at elucidating mechanisms of human disease. The GCRC can provide the necessary infrastructure and critical intellectual mass to reach a deeper understanding of human biology and to bring better health to persons in both developed and developing countries. A well-organized GCRC program should encompass educational as well as scientific components. It must be developed with an appropriate quality control infrastructure to provide physician-scientists a broad base of training in ethical principles in conduct of research and in scientific design and modern technology. The Human Genome Project has brought unprecedented opportunities to clinical investigators, but taking full advantage of them will require a rebirth of the GCRC as a program capitalizing on local strengths and differences, a more multicentric program, and a program reaching outside the GCRC walls, beyond institutional walls, and into the community. The GCRCs can appropriately be the clinical arm of the human genome project, harnessing the genetic revolution for human health. The GCRC model can provide a conceptual base on which new international patient-oriented research infrastructures can develop.

NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization.

Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer.

A model of troponin-I in complex with troponin-C using hybrid experimental data: the inhibitory region is a beta-hairpin.

We present a model for the skeletal muscle troponin-C (TnC)/troponin-I (TnI) interaction, a critical molecular switch that is responsible for calcium-dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin-C in complex with troponin-I, or in the ternary troponin-complex, have not yet delivered a high-resolution structure. Many groups have pursued different experimental strategies, such as X-ray crystallography, NMR, small-angle scattering, chemical cross-linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left-handed sense (Model L), and another that winds about TnC in a right-handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross-linking and FRET data. The inhibitory region TnI(95-114) is modeled as a flexible beta-hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a beta-hairpin region of the actin-binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the beta-hairpin is an important structural motif that communicates the Ca2+-activated troponin regulatory signal to actin.

Modulatory effects of endogenous adenosine on epinephrine secretion from the adrenal medulla of the rat.

The purpose of this study was to examine (1) whether endogenous adenosine receptors inhibit the release of epinephrine and norepinephrine from adrenal medulla in response to physiological and pharmacological stimuli and (2) whether the renin-angiotensin system modulates this effect of endogenous adenosine. We used a conscious animal model to approximate normal physiological conditions. Male Sprague-Dawley rats were treated with a surface adenosine receptor antagonist, 1,3-dipropyl- 8-(p-sulfophenyl)xanthine (DPSPX) to explore the effect of endogenous adenosine. Plasma epinephrine and norepinephrine levels in response to hydralazine-induced hypotension were measured in these animals. The same protocol was repeated in rats pretreated with either adrenalectomy or captopril. The results showed that DPSPX treatment significantly increased plasma epinephrine and norepinephrine levels at both baseline conditions and after hydralazine-induced hypotension. The results from the adrenalectomized rats showed that the difference in plasma epinephrine level between the control and DPSPX groups originated from the adrenal medulla. Pretreatment with captopril attenuated the rise of plasma epinephrine and norepinephrine levels in DPSPX-treated animals. This result suggests that endogenous adenosine receptors inhibit epinephrine release from the adrenal medulla and suppress plasma norepinephrine levels. When catecholamine release was stimulated by physiological and pharmacological stimuli, this inhibitory function of adenosine receptors was augmented. The renin-angiotensin system is at least partially responsible for the modulatory function of endogenous adenosine on the catecholamine response as demonstrated in this study.

Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats.

Nitric oxide (NO) is an endogenously synthesized effector molecule that acts as a neurotransmitter with novel properties in both the central and peripheral nervous systems. We previously reported that NO was involved in central cardiovascular regulation and modulated the baroreflex in the nucleus tractus solitarii (NTS) of rats. The aim of the present study was to determine whether NO and excitatory amino acids reciprocally release each other in the NTS. In normotensive Sprague-Dawley rats, intra-NTS microinjection of L-arginine (1 to 100 nmol/60 nL) produced a dose-dependent decrease in blood pressure and heart rate. Microinjection of excitatory amino acids L-glutamate and NMDA also produced depressor and bradycardic effects. These effects of L-glutamate or NMDA were blocked by prior administration of NO synthase inhibitor N(G)-methyl-L-arginine or N(G)-nitro-L-arginine methyl ester. Similarly, prior administration of N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly attenuated the depressor and bradycardic effect of L-arginine. These results demonstrated a reciprocal attenuation of NO synthase inhibitor and NMDA receptor antagonist on NMDA and L-arginine responses, respectively, in the NTS and suggest that NO and NMDA receptors may interact in central cardiovascular regulation.

Attenuated cardiovascular response to adenosine in the brain stem nuclei of spontaneously hypertensive rats.

We previously reported that adenosine has significant depressor effects in the nucleus tractus solitarii and area postrema of the rat. The purpose of this study was to determine whether the spontaneously hypertensive rat (SHR) has abnormalities in medullary sensitivity to adenosine. Male SHR and Wistar-Kyoto (WKY) rats (aged 12 to 15 weeks) were anesthetized with urethane, and blood pressure was monitored intraarterially. Stereotaxic microinjection (60 nL) of adenosine was made into the nucleus tractus solitarii and the area postrema and was confirmed histologically. Dose-related decreases in mean blood pressure and heart rate occurred in both strains tested, and this effect was completely abolished by 1,3-dipropyl- 8-p-sulfophenylxanthine (0.92 nmol), a potent adenosine receptor antagonist. However, there were significant differences between SHR and WKY rats in the magnitude of blood pressure and heart rate depression. A similar pattern of response was found in the area postrema. Thus, adenosine is a potent depressor agent in the nucleus tractus solitarii and area postrema of rats, and adenosine has significantly fewer depressor effects in SHR. These data suggest that alterations in purinergic mechanisms of central cardiovascular control exist in the SHR model.

Cardiovascular effects of nitric oxide in the brain stem nuclei of rats.

Nitric oxide, synthesized from the semiessential amino acid L-arginine by nitric oxide synthase, is a remarkable regulatory molecule and plays an important role in physiological functions. However, the physiological role of nitric oxide in cardiovascular regulation by the central nervous system is not well understood. In this study we investigated the cardiovascular effects of nitric oxide in the lateral ventricle, nucleus tractus solitarii, area postrema, and rostral ventrolateral medulla in urethane-anesthetized male Sprague-Dawley rats. Microinjection of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, into the cerebral ventricle of rats elicited a dose-dependent increase in blood pressure and heart rate. This suggests that nitric oxide may be involved in central cardiovascular regulation. Unilateral microinjection (60 nL) of L-arginine (1 to 100 nmol) into the nucleus tractus solitarii and rostral ventrolateral medulla produced prominent dose-related depressor and bradycardic effects and reduced renal sympathetic nerve activity. However, L-arginine had no significant cardiovascular effects in the area postrema. In addition, 4 to 6 hours after intravenous injection of bacterial endotoxin-lipopolysaccharide (10 mg/kg), there was a time-related potentiation of the L-arginine-induced depressor and bradycardic effects in the nucleus tractus solitarii. These results indicate that nitric oxide is involved in central cardiovascular regulation. The depressor effect of nitric oxide in the nucleus tractus solitarii and rostral ventrolateral medulla may be through inhibition of renal sympathetic nerve activity.

Antihypertensive metabolites of alpha-methyldopa.

alpha-Methyldopa acts through a metabolite in the central nervous system. Of the three metabolites most prominently considered as potential mediators of alpha-methyldopa hypotension--alpha-methyldopamine, alpha-methylnorepinephrine (MNE), and alpha-methylepinephrine (ME)--ME is the most potent depressor agent following intravenous infusion into rats, following injection into the lateral ventricle, and following injection into the solitary tract nucleus (NTS). The depressor effect of ME in the NTS is attenuated as effectively by timolol as by yohimbine, while the combination of both timolol and yohimbine completely abolishes the pharmacological activity of ME in the NTS. ME is approximately eightfold more potent than MNE in the NTS and also has a greater susceptibility to timolol attenuation.

Characterization of the distribution of potential short restriction fragments in nucleic acid sequence databases. Implications for an alternative to chemical synthesis of oligonucleotides.

We have searched the GenBank nucleic acid sequence database for potential short restriction fragments. All possible oligonucleotides up to length five are found at least once flanked by known restriction recognition patterns. Thus, searches in the database for a specific sequence corresponding to a desired oligonucleotide would often point to one or more sources of short, retrievable fragments containing that sequence. These results underscore the potential of nucleic acid sequence databases in planning experiments.

Immunohistochemical study of catecholamine enzymes and neuropeptide Y (NPY) in the rostral ventrolateral medulla and bulbospinal projection.

The purpose of this study was to determine whether neuropeptide Y (NPY) terminals in the intermediolateral spinal cord originate from the rostral ventrolateral medulla (RVLM). Immunohistochemical staining of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and NPY in the rat brainstem and spinal cord were performed in this study in order to examine consequences of lesions of the RVLM and of intracisternal injections of 6-hydroxydopamine (6-OHDA) on catecholamine and NPY immunoreactivity in the intermediolateral column (IML) of rats. In addition, ricin, a retrograde neurotoxin, was applied in the superior cervical ganglion (SCG) to determine its effect on catecholamine and NPY immunoreactivity in the IML. Computer-aided image analysis was used to quantify the immunohistochemical changes in the RVLM and spinal cord. The results demonstrated that many catecholamine- and NPY-containing neurons and/or fibers existed in the RVLM and their terminals were found in the IML. After administration of 6-OHDA intracisternally, the catecholamine and NPY immunoreactivities were decreased both in the brainstem and IML of the spinal cord. Following unilateral microinjection of 6-OHDA into the RVLM, the number of NPY- and catecholamine-containing neurons decreased and there was a reduction in neuron terminals on the ipsilateral side. After injection of ricin into the SCG, the catecholamine and NPY neurons of the medulla were not affected, whereas their terminals in the IML decreased ipsilaterally. These results indicate that most of the catecholamine- and NPY-immunoreactive terminals found in the IML originated in the RVLM. These terminals appear to project towards the superior cervical ganglia.

Caffeine and hypertension.

The effect of prolonged caffeine administration on blood pressure in hypertensive subjects was assessed in a double-blind placebo-controlled study. Eighteen hypertensive subjects participated, nine of whom received placebo throughout the study and nine of whom received placebo during the first three days, caffeine during the subsequent seven days, and placebo during the final four days of the two-week study. Those who received caffeine were given 250 mg with meals three times daily. There were no untoward reactions in the course of the study, but one subject with unacceptably high blood pressures while receiving placebo had to be discharged from the study to resume antihypertensive therapy. Systolic blood pressure was immediately increased (9.2 +/- 3.4 mm Hg) within 15 minutes after the first dose of 250 mg of caffeine. On the first day of caffeine, systolic pressure was increased a mean of 7.3 +/- 4.0 mm Hg, but this was no longer significant after the initial day of caffeine administration. Diastolic pressure showed a trend toward increasing, but this never reached significance. The minor increases in plasma catecholamine levels and plasma renin activity were not significant on either a short- or long-term basis. After discontinuation of caffeine, no overshoot phenomena were observed. It is concluded that prolonged administration of caffeine is not associated with significant elevation in blood pressure, plasma catecholamine levels, or plasma renin activity in patients with borderline hypertension.

Evidence that 5-HT(2) Antagonism Elicits a 5-HT(3)-Mediated Increase in Dopamine Transmission.

Amperozide, a novel atypical antipsychotic drug with few extrapyramidal side effects, is a strong serotonin(2) (5-HT(2)) antagonist but has low affinity for dopamine receptors in vitro. The effect of amperozide on the dopaminergic synapse was studied with an in vivo microdialysis technique using anesthetized male Sprague-Dawley rats. Following implantation of dialysis probes into the striatum and nucleus accumbens (NuAc), amperozide was intravenously infused as six consecutive incremental doses (0.5, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/kg) at intervals of 15 min. From the beginning of drug infusion, perfusates were collected in fractions every 30 min throughout a total period of 120 min. The samples were then immediately analyzed by high-performance liquid chromatography with electrochemical detection. Amperozide induced a dose-related elevation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels in both areas. p-Chlorophenylalanine (pCPA) pretreatment abolished the production of 5-HIAA in both areas and attenuated the amperozide-induced rise of DOPAC but not of dopamine. After pretreatment with an intravenous 5-HT(3) antagonist, MDL 72222, the amperozide-induced changes in dopamine, DOPAC and 5-HIAA in both areas were lower than in the saline control group. Preliminary data showed that after pCPA pretreatment, incremental concentrations of the 5-HT(3) agonist 1-(m-chlorophenyl)-biguanide perfused via the probe also produced significant elevation of dopamine and DOPAC levels in these two areas. Taken together, these results suggest that amperozide may directly block 5-HT(2) receptors in the striatum and NuAc, thereby enhancing 5-HT transmission. The enhanced 5-HT transmission may activate postsynaptic 5-HT(3) receptors located on the dopaminergic terminals, leading to changes in dopamine transmission in these two areas. Copyright 1995 S. Karger AG, Basel

Nicotinamide attenuates methamphetamine-induced striatal dopamine depletion in rats.

The aim of the present study was to examine the effects of nicotinamide, a co-factor in the electron transport chain, on the relationship between methamphetamine (MA)-induced striatal dopamine (DA) depletion and energy metabolism change. Four injections of MA (10 mg/kg, i.p.) at 2 h intervals resulted in decreases of 51% and 23%, respectively, in striatal DA and adenosine 5'-triphosphate (ATP) levels 5 days later. Nicotinamide (500 mg/kg, i.p.) treatment prior to each MA injection attenuated the reductions of striatal DA and ATP contents. Nicotinamide had no long-term effects on striatal DA and ATP levels. These findings suggest that energy impairment might play a role in MA-induced DAergic neurotoxicity in the striatum.

Endothelin-3 reduces C-type natriuretic peptide-induced cyclic GMP formation in C6 glioma cells.

The effect of endothelin-3 (ET-3) on C-type natriuretic peptide (CNP)-induced guanosine 3',5'-cyclic monophosphate (cGMP) was examined in C6 glioma cells, CNP-induced cGMP formation was both time- and dose-dependent, with an EC50 value of about 10 nM. While ET-3 and phorbol 12-myristate 13-acetate (PMA) had no effect on basal cGMP production, both compounds were potent inhibitors of CNP-induced cGMP formation, with IC50 values of approximately 10 and 2 nM, respectively. Although protein kinase C (PKC) inhibitors had no effect on basal cGMP formation, Ro 31-8220, a PKC inhibitor, reversed the ET-3 inhibition on CNP-induced cGMP formation by 63% and that of PMA almost completely. Our findings suggest that stimulation of cGMP formation by CNP in C6 glioma cells is negatively modulated by PKC activation, and that the inhibitory action of ET-3 on CNP-stimulated cGMP formation is mediated partly by PKC.