Metastasis suppressor genes: a role for raf kinase inhibitor protein (RKIP)

The metastatic cascade is a complicated process that involves many steps from gain of the metastatic phenotype in the primary tumor cells through establishment of macroscopic tumor at the distant target organ. A group of genes, termed metastasis suppressor genes (MSG), encode for proteins that inhibit various steps of the metastatic cascade. Accordingly, loss of MSG promotes the metastatic phenotype. Although several MSG have been identified, the mechanisms through which they enhance metastasis are not clearly defined. Gene array analysis of a low metastatic LNCaP prostate cancer cell line compared to its highly metastatic derivative C4-2B prostate cancer cell line revealed decreased expression of raf kinase inhibitor protein (RKIP) in the C4-2B cell line. RKIP blocks the activation of several signaling pathways including MEK, G-proteins and NFkappaB. Immunohistochemical analysis of prostate cancer primary tumors and metastases revealed that RKIP protein expression was decreased in metastases. Restoration of RKIP expression in the C4-2B cell line diminished metastasis in a murine model. These results demonstrate that RKIP is a MSG. Loss of RKIP enhanced both angiogenesis and vascular invasion, and protected against apoptosis. These findings suggest that targeting the RKIP pathway may diminish the metastatic cascade. However, challenges exist as to the best method to target RKIP expression. Restoration of RKIP expression in all cancer cells in vivo is challenging. A plausible strategy is to use small molecules that target proteins in signaling pathways that are dysregulated due to loss of RKIP.     

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide-cyclic GMP pathway

Fencamfamine (FCF) is an indirect dopamine agent with effects similar to amphetamine and cocaine. In the present study, we investigate changes in Na,K-ATPase, cyclic AMP-dependent protein kinase (PKA) and nitric oxide synthase (NOS) activity and cyclic GMP levels in the nucleus accumbens (NAc) and striatum (ST) of animals acutely or repeatedly treated with FCF (3.5 mg/kg). Na,K-ATPase had a similar activity in control and repeatedly treated animals, but was reduced in the NAc of the acute group. This enzyme was reduced in the ST in acute and repeatedly treated animals, compared to the control group. Expression of the _1,2,3-Na,K-ATPase isoforms in the NAc and the ST was not altered in all groups studied. Acute FCF induced a significant increase in PKA activity in both the ST and the NAc. Repeatedly treated animals showed a higher increase in PKA activity in the NAc, but not in the ST, when compared to the acute group. There was also an increase in both NOS activity and cyclic GMP levels only in the NAc of FCF repeatedly treated animals compared to the acute and control groups. We suggest that chronic FCF treatment is linked to a modification in Na,K-ATPase activity through the PKA and NO–cyclic GMP pathway.     

A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition

The firefly luciferin-luciferase reaction has been used to set up an assay for protein kinase based on measuring ATP consumption rate as the first-order rate constant for the kinase reaction. The assay obviates the problems encountered with previous bioluminescent protein kinase assays such as interference with the luciferase reaction from library compounds, nonlinear standard curves, and limited dynamic ranges. In the assay described in the present paper luciferase and luciferin are present during the entire kinase reaction, and the light emission can be measured continuously. In an HTS situation the light emission is measured only twice, i.e., initially and after a predetermined time. After a fivefold reduction of the ATP concentration a Z' value of 0.96 was obtained. Light emission data from samples with kinase are normalized with light emission data from blanks without kinase. First-order rate constants for the kinase reaction calculated from normalized light emission are not affected by a moderate degree of inactivation of luciferase and luciferin during the measuring time. The constants have the same value at all ATP concentrations much lower than the K(m) of the luciferase and the kinase. These factors make the assay very robust and influenced neither by ATP concentration nor by luciferase inhibition. The measuring time depends on the kinase activity and can be varied from minutes to more than 8 h provided the kinase is stable and the evaporation of water from the wells is acceptable. The assay is linear with respect to kinase activity over three orders of magnitude. The new reagents also allowed us to determine K(m) values for ATP and for Kemptide.     

Potentiation of cyclic AMP and cyclic GMP accumulation by p38 mitogen-activated protein kinase (p38MAPK) inhibitors in rat pinealocytes.

The effects of p38 mitogen-activated protein kinase (p38MAPK) inhibitors on the adrenergic-stimulated cyclic nucleotide production in rat pinealocytes were investigated. Treatment with SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)IH-imidazole] and SB203580 [4-(4-fluoropheny)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole] (1-100 microM), two pyridinyl imidazole compounds that inhibit p38MAPK, as well as SB202474 [4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)IH-imidazole], an inactive analog, was effective in potentiating norepinephrine- and isoproterenol-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation in a concentration-dependent manner. All three compounds caused a greater increase in the cGMP than the cAMP response, with SB202474 being substantially more potent than the two active analogs. At 100 microM, SB202474 potentiated the isoproterenol-stimulated cAMP and cGMP accumulation by 65 and 500%, respectively. Pharmacological studies indicated that the potentiating effect of SB202474 was independent of protein kinase C activation, intracellular calcium elevation, or serine/threonine phosphatase inhibition, three pathways known to potentiate the beta-adrenergic-stimulated cyclic nucleotide responses in rat pinealocytes. In contrast, the potentiating effect of SB202474 was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine. At 100 microM, all three compounds inhibited cAMP- and cGMP-phosphodiesterase activities by 50 and 80%, respectively. These results suggest that the commonly used p38MAPK inhibitors can modulate cyclic nucleotide responses through phosphodiesterase inhibition, a mechanism that appears to be independent of p38MAPK inhibition.     

Selective inhibition of a cyclic nucleotide independent protein kinase (G type casein kinase) by quercetin and related polyphenols

The effect of quercetin and a number of structurally related phenolic compounds upon the activity of three different purified protein kinases was examined. Whereas the catalytic subunit of a cyclic AMP-dependent protein kinase and an A type (using only ATP) cyclic nucleotide-independent casein kinase (CKA) were not affected, a G type (using GTP as well as ATP) casein kinase (CKG) was selectively inhibited by several bioflavonoid structures. Kinetic studies showed that quercetin behaved as a competitive inhibitor toward the nucleotidic substrate and exhibited a high affinity for the ATP (Ki = 0.75 microM) and GTP (Ki = 0.22 microM) site of the enzyme. Considering the CKG inhibitory potency of a series of flavonoid, cinnamic acid and coumarin derivatives, it is suggested that the biological activity lays upon a common structural feature involving a phenolic ring bearing a side chain with conjugated double bonds and an oxygenated function, as found in the coumaroyl residue. These observations suggest that quercetin and related compounds may lead to a shift in intracellular protein phosphorylations by selectively inhibiting a particular type of protein kinase activity (CKG). It remains to be established whether this process may contribute to the mechanism of action of flavonoids upon cellular metabolism, particularly in the case of malignant cells.     

Modification of cholinergically induced convulsive activity and cyclic GMP levels in the CNS

Soman, an organophosphorus cholinesterase inhibitor, was used to produce cholinergic stimulation and convulsions in rats. Soman (1 or 2 × LD₅₀) elevated cyclic 3′5′ guanosine monophosphate (cGMP) levels in the cerebellum, before the onset of cholinergic symptoms, during the stage prior to convulsions, at doses which produced no convulsive activity (1/2LD₅₀) and greatly elevated cGMP levels at the onset and during convulsions. Pretreatment which reduced the convulsive activity of soman also attenuated the increase in cGMP in the cerebellum.The results indicated that soman-induced convulsions and the resulting increase in cerebellar cGMP concentrations could not be related solely to increased cholinergic activity or to muscarinic effects. Pretreatment of poisoned animals with antimusarinic or antinicotinic compounds or atropine plus an oxime (HI-6) which protected animals from the lethal effects of soman, had little effect on severity of convulsions or cGMP levels. On the other hand, both parameters were sensitive to anticonvulsants (i.e. clonazepam) which, are not thought to produce their effects via anticholinergic actions. In addition, intracerebroventricular injections of dibutyryl cGMP (dbcGMP) caused convulsions which were blocked by small doses of clonazepam but were only slightly affected by atropine or mecamylamine, given alone.Stimulation of climbing fibres to the cerebellum may result in the release of an excitatory amino acid which may subsequently cause elevation of cGMP. However, soman failed to alter cerebellar concentrations of putative excitatory or inhibitory neurotransmitters while destruction of these fibres with 3-acetylpyridine prior to injection of soman failed to block either the convulsions or the increase in cGMP concentrations.These results suggest that cGMP may play a role in the initiation and continuation of soman-induced convulsions which were not related to specific cholinergic receptors but to general neuronal excitation. The results also indicate the involvement of non-cholinergic mechanisms in soman-induced convulsions, and in the rise in cerebellar cGMP levels; however, the identity of these mechanisms was not known.     

Evidence for hypothalamic K+(ATP) channels in the modulation of glucose homeostasis

Several lines of evidence support the hypothesis that ATP-sensitive K+ channels (K+(ATP)) participate in the brain's regulation of peripheral glucose homeostasis. In testing this hypothesis we conducted a series of in vivo experiments using albino rats and bilateral intrahypothalamic injections of K+(ATP) channel blockers, glibenclamide and repaglinide. The results show that 0.2 and 2.0 nM injections of glibenclamide lowered blood glucose in a dose-dependent manner. During mild insulin-induced hypoglycemia, hypothalamic glibenclamide delayed recovery to normoglycemia. The impaired recovery was associated with a reduction in plasma norepinephrine (P<0.001), though circulating epinephrine and glucagon were not reduced. In a separate experiment, 2-deoxy-D-glucose (200 mg/kg) was intraperitoneally administered to produce neuroglucopenia. Hypothalamic injections of either glibenclamide or repaglinide significantly blunted compensatory hyperglycemic responses (P<0.01). In a feeding study, 2.0, but not 0.2 nM of hypothalamic glibenclamide, reduced chow intake over a 2-h period (P<0.01). The results support the hypothesis that hypothalamic K+(ATP) channels participate in central glucose-sensing and glucose regulation.     

Evidence for regulation of interaction of endogenous protein kinase C (PKC) substrates with plasma membrane by PKC down-regulation in K562 cells

In the particulate fraction obtained from PKC-down regulated K562 cells by treatment for 24 h with 200 nM TPA, phosphorylation of two proteins with molecular weight, 100 kDa and 23 kDa (designated p100 and p23, respectively) was depleted and addition of exogenous purified PKC to this fraction failed to restore their phosphorylation. However, in the soluble fraction, all of phosphoproteins abolished by long-term treatment with TPA were restored by exogenously added PKC. Phosphorylation of two proteins was increased by short-term treatment (20 min), and diminished with the persistant exposure to TPA as well as at a concentration as low as 1 nM. When K562 cells were treated with 1 nM and 200 nM TPA for 24 h, phosphorylation of p100 was restored with or without exogenous PKC on 2–3 day and 6 day after removal of treated TPA, respectively. Two-dimensional electrophoresis of phosphoproteins revealed that phosphorylated p100 (pl=5.9) and a p66 species were completely absent from the particulate fraction of K562 cells treated with 200 nM TPA for 24 h. These results suggest that the interaction of sensitive endogenous substrates, p100 and p23 with the plasma membrane might be regulated by PKC-down regulation without their displacement to the cytosol and the interaction of p100 with the membrane might be reversible.     

The role of Raf kinase inhibitor protein (RKIP) in health and disease

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding protein (PEBP) family. RKIP plays a pivotal modulatory role in several protein kinase signaling cascades. RKIP binds inhibits Raf-1-mediated phosphorylation of MEK through binding to Raf-1. Protein kinase C (PKC) phosphorylates RKIP, resulting in release of Raf-1 and activation of MEK and ERK. The phosphorylated RKIP binds to and inhibits G-protein-coupled receptor kinase, resulting in sustained G-protein signaling. The regulatory role that RKIP has in cell signaling is reflected in its role in physiology and pathophysiology. RKIP is involved in neural development, cardiac function and spermatogenesis and appears to have serine protease activity. In addition to its roles in physiology, dysregulated RKIP expression has the potential to contribute to pathophysiological processes including Alzheimer's disease and diabetic nephropathy. RKIP has been shown to fit the criteria of being a metastasis suppressor gene, including having decreased expression in prostate cancer metastases and restoring RKIP expression in a prostate cancer cell line diminishes metastasis in a murine model. Clearly, RKIP has multiple molecular and cellular functions. In this review, RKIP's molecular roles in intracellular signaling, its physiological functions and its role in disease are described.     

The role of protein kinase C-alpha (PKC-alpha) in cancer and its modulation by the novel PKC-alpha-specific inhibitor aprinocarsen

As our understanding of tumorigenesis increases, interference with the various signaling pathways of tumor cells has become an attractive approach to arresting tumor cell growth and overcoming chemoresistance. Among many intracellular signaling proteins, protein kinase C (PKC) isoenzymes have been identified as possible targets to render tumor cells more susceptible to apoptosis and growth arrest. We review the known biology of the alpha-isoenzyme of PKC in different cancers to provide a rational approach for developing targeted therapies using PKC modulators, including aprinocarsen, an antisense oligonucleotide (ASO) against PKC-alpha.     

Essential role of L-arginine uptake and protein tyrosine kinase activity for NO-dependent vasorelaxation induced by stretch, isometric tension and cyclic AMP in rat pulmonary arteries

1. The NO-dependent component of cyclic AMP-induced vasorelaxation in rat pulmonary arteries is critically dependent on extracellular L-arginine but independent of endothelial cell intracellular [Ca(2+)]. We examined whether L-arginine uptake was also essential for NO production induced by passive stretch or isometric tension, processes also reported to be Ca(2+)-independent. 2. The passive length-tension curve was depressed by physiological concentrations of L-arginine (400 microM; P<0.05). Inhibition of the y(+) transporter with 10 mM L-lysine, NO synthase with L-NAME (100 microM), or protein tyrosine kinase with erbstatin A (30 microM) caused identical upward shifts (P<0.001), alone or in combination. Tyrphostin 23 was similar to erbstatin A, whilst the inactive analogue tyrphostin A1 and genistein were without effect. 3. L-arginine (400 microM) shifted the PGF(2 alpha) concentration-response curve under isometric conditions to the right (P<0.05), whereas L-NAME or L-lysine caused a leftward shift (P<0.001). Tyrphostin 23 (30 microM) more than reversed the L-arginine-induced suppression of PGF(2 alpha)-induced tension; subsequent addition of L-NAME had no effect. The L-lysine-sensitive component of CPT cyclic AMP-induced vasorelaxation was abolished by erbstatin A. 4. ACh-induced vasorelaxation was approximately 80% inhibited by L-NAME, but was not affected by L-lysine or 400 microM L-arginine. Erbstatin A reduced the vasorelaxation by only approximately 25%. 5. We conclude that activation of NO production by stretch, isometric tension, or cyclic AMP in rat pulmonary arteries is critically dependent on the presence and uptake of physiological concentrations of extracellular L-arginine, and protein tyrosine kinase activity. This directly contrasts with ACh-induced vasorelaxation, which was independent of extracellular L-arginine, and relatively unaffected by tyrosine kinase inhibition.     

Evidence for the involvement of cyclic GMP in adenosine-induced, age-dependent vasodilatation.

1. Adenosine-induced dilatation of rat aorta was present in aorta taken from 4 week-old rats, attenuated with increase in age of rats to 8 weeks, and was virtually absent in the aorta from 12 week-old rats. 2. Removal of the endothelium by mechanical rubbing attenuated adenosine-induced dilatation. 3. Haemoglobin and methylene blue partly reversed the adenosine-induced endothelium-dependent dilatation. 4. The order of potency of adenosine derivatives was 5'-(N-ethylcarboxamido)adenosine (NECA) greater than 2-phenylaminoadenosine (CV-1808) greater than 2-chloroadenosine greater than N6-([R]-[-]-phenylisopropyl)adenosine (R-PIA) greater than adenosine greater than N6-cyclohexyladenosine (CHA) greater than N6-([S]-[+]-phenylisopropyl)adenosine (S-PIA), indicating that adenosine receptors mediating the dilatation are of the A2 subtype. 5. [3H]-NECA bound to preparations of membranes from rats of 4 weeks old; it was displaced more effectively by NECA and the A2 ligand CV-1808 than by the A1 ligands CHA and S-PIA. ligands CHA and S-PIA. 6. The number but not affinity of specific binding sites for NECA decreased considerably with increase in age of rats to 8 weeks, and binding sites for [3H]-NECA were hardly detected in membrane preparations from rats of 20 weeks old. 7. Adenosine caused a marked increase in cyclic GMP production, but did not induce an increase in the cyclic AMP level. 8. This increase in cyclic GMP production induced by adenosine was abolished by methylene blue or 8-phenyltheophylline, or by removal of the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP

We investigated the roles of cyclic GMP and cyclic AMP in the inhibition of rabbit platelet aggregation and degranulation by two nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1; the active metabolite of molsidomine), with particular reference to the synergistic interaction of these drugs with prostaglandin E1 (PGE1). Changes in platelet cyclic [3H]GMP and cyclic [3H]AMP were measured by rapid and sensitive prelabeling techniques, the validity of which were confirmed by radioimmunoassays. Incubation of the platelets with 0.1 to 10 microM SNP alone for 0.5 min caused progressively greater inhibitions of platelet function associated with large dose-dependent increases in cyclic [3H]GMP and 1.4- to 3.0-fold increases in cyclic [3H]AMP. However, addition of SNP with the adenylate cyclase activator, PGE1, at a concentration of the latter that had little effect alone, caused much larger increases in cyclic [3H]AMP and greatly enhanced the inhibition of platelet aggregation. SIN-1 had effects similar to those of SNP, although it was less active. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) diminished the increases in cyclic [3H]AMP caused by SNP or SIN-1 in both the presence and absence of PGE1 but reduced the inhibition of platelet function caused by the nitrovasodilators only in the presence of PGE1. These results suggest that, although cyclic GMP may mediate the inhibition of rabbit platelet function by high concentrations of nitrovasodilators added alone, the synergistic interaction of lower concentrations with PGE1 depends on an enhanced accumulation of cyclic AMP. Synergistic effects on cyclic [3H]AMP accumulation were also observed on incubation of platelets with SNP and adenosine, another activator of adenylate cyclase. Hemoglobin, which binds nitric oxide, blocked or reversed the increases in both cyclic [3H]GMP and cyclic [3H]AMP in platelets caused by the nitrovasodilators added either alone or with PGE1. Cilostamide, a selective inhibitor of platelet low Km cyclic AMP phosphodiesterase, had effects on platelet cyclic [3H]AMP accumulation identical to those of SNP, suggesting that the action of the latter depends on inhibition of the same enzyme. M&B 22,948, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in both cyclic [3H]GMP and cyclic [3H]AMP caused by SNP. A hyperbolic relationship was found between the increases in cyclic [3H]GMP and cyclic [3H]AMP caused by different concentrations of SNP; this relationship was not affected by addition of M&B 22,948. The results strongly suggest that the increases in platelet cyclic [3H]AMP caused by nitrovasodilators in the presence or absence of activators of adenylate cyclase are mediated by the inhibition by cyclic GMP of cyclic AMP breakdown.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that relaxation of hog biliary muscle is mediated by the interaction between the protein inhibitor of cyclic AMP dependent protein kinase and cholecystokinin C-terminal peptides

The interaction of various cholecystokinin (CCK) peptides with the protein inhibitor (PK-I) of cyclic AMP dependent protein kinase (A-PK) has been studied. The order of the affinities (ED50) for relaxation of hog biliary muscle by a series of C-terminal peptides of CCK correlated with the order of the potencies for A-PK-catalyzed phosphorylation of the sarcoplasmic reticulum enriched fraction (SR-F) in the muscle. CCK-4 peptide inhibited the PK-I of A-PK. The apparent Km value of the peptide was 11.5 microM. Scatchard plot analysis for the binding of [14C]CCK-6 peptide to the PK-I showed a dissociation constant (Kd) of 11.2 microM. These results indicated that the Kd value agreed with the ED50 value of CCK-6 and the Km value of CCK-4. It is proposed that a receptor for CCK C-terminal peptides is probably a component in the PK-I; binding of the peptides to this site usually results in the relaxation by these allosteric inhibitors of the PK-I.     

Enhanced nitric oxide and cyclic GMP formation plays a role in the anti-platelet activity of simvastatin

BACKGROUND AND PURPOSE: It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin. EXPERIMENTAL APPROACH: After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately. KEY RESULTS: Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC(50) range of 52-158 microM. We also demonstrated that simvastatin (20-80 microM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64+/-0.12 to 3.52+/-0.21-5.10+/-0.14 micromol min(-1) mg protein(-1)) and guanylyl cyclase activity (from 142.9+/-7.2 to 163.5+/-17.5-283.8+/-19.5 pmol min(-1) mg protein(-1)) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin.     

Protective effect of a protein kinase inhibitor on cellular injury induced by cephaloridine in the porcine kidney cell line LLC-PK(1)

We investigated the effects of a protein kinase C inhibitor and a tyrosine kinase inhibitor on the cellular injury induced by cephaloridine in an established renal epithelial cell line, LLC-PK(1). Cephaloridine increased the leakage of lactate dehydrogenase (LDH) from LLC-PK(1) cells into the medium and also caused an increase in the level of lipid peroxide (index of oxidative stress) in the cells. Treatment of the cells with a hydroxyl radical scavenger, dimethylthiourea (DMTU), inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. A protein kinase C inhibitor, H-7, and tyrosine kinase inhibitors, genistein and lavendustinA, inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. These results suggest that a signaling pathway which involves protein kinase C and tyrosine kinase plays a role in the generation of reactive oxygen species in LLC-PK(1) cells damaged by cephaloridine.     

A role for cyclic GMP in the initiation of cardiac pressor reflexes by bradykinin and capsaicin

Reflexogenic effects of bradykinin on the cardiac sympathetic afferents supplying the dog epicardium were inhibited by treatment of the heart surface with either methylene blue or the compound LY-83583 (6-anilino-5,8-quinolinedione), which are known to lower cellular levels of cyclic GMP. Stimulation of cyclic GMP production by epicardial treatment with the NO-containing synonimine compound, SIN-1, potentiated bradykinin-induced reflex effects. In contrast, the sympathetic cardiac pressor response induced by capsaicin was not influenced by epicardial treatment with either methylene blue, LY-83583 or SIN-1. These findings indicate the importance of cyclic GMP for the reflexogenic activity of bradykinin, but not that of capsaicin on the sympathetic afferents supplying the dog heart.     

Isoprenaline-induced changes in activity of the endogenous inhibitor of cAMP-dependent protein kinases under conditions of beta-adrenoceptor supersensitivity

Isoprenaline-induced changes in activity of an endogenous, specific inhibitor of cAMP-dependent protein kinases (type I inhibitor) under conditions of experimental supersensitivity of beta-adrenoceptors were investigated in rat hippocampus and brain stem. Both subchronic administration of reserpine (2.5 mg kg-1, i.p., 4 days, once daily) and i.c.v injections of 6-hydroxydopamine (250 micrograms/ventricle, bilaterally, 48 h apart), which are known to increase beta-adrenoceptor sensitivity in the rat brain, markedly enhanced the response of the type I inhibitor activity to isoprenaline. To obtain a significant decrease of type I inhibitor activity in the examined brain structures of these animals, doses of isoprenaline 2-5 times lower than in control groups had to be used. It is suggested that the isoprenaline-induced decrease of type I inhibitor activity might be used as an index of central beta-adrenoceptor reactivity in-vivo.