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J E Wikberg

Publications and source records attributed to J E Wikberg.

At least 127 records · Page 7Linked to original sources
PubMed1989

Purification of soluble guanylate cyclase enzyme from human platelets.

Soluble guanylate cyclase enzyme was purified from human platelets. The soluble fraction of the lysed platelets was sequentially chromatographed over DEAE-sepharose, GTP-agarose and HPLC size-exclusion columns. About 0.1 mg of purified enzyme could be obtained from 2000 ml of platelet rich plasma. The purified enzyme had the specific activity of 205 nmoles cGMP/mg/min with Mn2+ as cofactor. The enzyme eluted at the 160,000 daltons position from the size-exclusion column. Electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions revealed two subunits of 83,000 and 71,000 daltons respectively.

Blood Platelets↗
PubMed1988

Relationship between forskolin and calcium-calmodulin stimulation of rat cerebral cortex adenylate cyclase: enzyme activation modulates substrate (MgATP) affinity.

In membranes prepared from the rat cerebral cortex EGTA 0.4-100 mumol/l was found to dose-dependently inhibit adenylate cyclase activity, both during basal conditions and when the cyclase activity had been stimulated with 10 mumol/l forskolin. Addition of calcium 2-30 mumol/l (in excess of EGTA) totally prevented the inhibition induced by EGTA, both in the absence and presence of forskolin. These data are consistent with the notion that in the absence of EGTA the rat cortex adenylate cyclase is considerably stimulated by endogenous calcium-calmodulin. The data also show that stimulation with endogenous calcium-calmodulin is more than additive with that of forskolin indicating an action on identical cyclase units. The kinetics of the rat cortex adenylate cyclase for its substrate (MgATP) was also investigated. Activation of the cyclase with endogenous calcium-calmodulin, induced a marked increase in the Vmax of the enzyme and a concomitant almost 2-fold increase in the Km. Stimulation with 10 microM forskolin also induced a marked increase in the Vmax as well as an almost 3-fold increase in the Km. A combination of the two stimulants caused a further increase in Vmax as well as Km; the Vmax being increased more than 17-fold and the Km being increased 3.5-fold over the basal. The data indicates that both calcium-calmodulin and forskolin induces a similar type of activation of the adenylate cyclase which is associated with a lowered affinity for its substrate.

Adenosine Triphosphate↗
PubMed1988

Inhibition of cyclic AMP production by alpha 2-adrenoceptor stimulation in the guinea-pig spinal cord slices.

In spinal cord slices isolated from guinea-pig and preincubated with 3H-adenine, 0.3-30 microM forskolin induced a dose-dependent increase in the content of 3H-cAMP, the maximal increase being about 8-fold. The selective alpha 2-adrenergic agonist UK-14,304 (10 microM) reduced both the basal and the forskolin stimulated levels of 3H-cAMP by 18-32%. Dose response curves of the effect of UK-14,304 on cAMP production in the spinal cord slices, stimulated with 3 microM forskolin, showed an IC50 of 37 nM and a maximally inhibitory effect of 27%. A number of other alpha 2-adrenergic agonist (clonidine, guanfacine, B-HT 920 and B-HT 933) also inhibited the forskolin stimulated 3H-cAMP production; clonidine and guanfacine being almost equipotent with UK-14,304, but their maximal inhibitory effects being only about 6-7%. B-HT 920 and B-HT 933 were less potent and their maximal inhibitory effects about 16-21%. The dose response curve of UK-14,304 on inhibition of forskolin stimulated cAMP production was shifted almost 50-fold to the right by 0.3 microM yohimbine. Prazosin (0.3 microM) did not affect the UK-14,304 dose response curve. It is concluded that alpha 2-adrenoceptor stimulation mediates inhibition of cAMP production in the guinea-pig spinal cord.

Adrenergic alpha-Agonists↗
PubMed1988

Receptors for insulin-like growth factor-I in plasma membranes isolated from bovine mesenteric arteries.

Binding of IGF-I to plasma membranes from bovine mesenteric arteries was studied. The maximal specific binding of IGF-I was found to be 7.4 +/- 1.7% of total 125I-IGF-I added to the incubation medium. Unlabelled IGF-I displaced 125I-IGF-I with an IC50 value of 0.5 nmol/l and a maximal displacement of 64.2 +/- 2.8% of total binding. The potency of insulin to displace 125I-IGF-I was 100-1000-fold lower. Cross-linking of 125I-IGF-I to the receptor with disuccinimidyl suberate, followed by SDS-polyacrylamide gel electrophoresis under reducing conditions showed an IGF-I binding protein with a molecular weight of 146,000 Dalton. In summary, we have shown the presence of receptors for IGF-I in plasma membranes isolated from macrovessels. The binding characteristics and the size of the binding unit were found to be similar to those of the IGF-I receptor found in cultured vascular smooth muscle cells. Furthermore, insulin at high concentrations was found to interact with the IGF-I receptor.

Animals↗
PubMed1988

Attomole sensitive radioimmunoassay for cyclic GMP.

The sensitivity of the radioimmunoassay for cyclic GMP has been improved to readily detect attomole (200-300) amounts of the nucleotide in tissue extracts. The improved sensitivity has been achieved by using a high specific radioactivity [125I] cyclic GMP 2'O-succinyl tyrosine methyl ester (2200 Ci/mmole) as labeled antigen in conjunction with acetylation of the cyclic GMP in the samples as well as by using a selected cyclic GMP antibody. The high specific [125I] cyclic GMP 2'O-succinyl tyrosine methyl ester was prepared radiochemically pure by radioiodination of cyclic GMP 2'O-succinyl tyrosine methyl ester and purification using HPLC. The new attomole sensitive cyclic GMP radioimmunoassay is simple to perform and fast and has been applied with success to determine attomole quantities of cyclic GMP in cultured smooth muscle cells and AG 1523 fibroblasts.

Animals↗
PubMed1987

Spinal cord alpha 2-adrenoceptors may be located postsynaptically with respect to primary sensory neurons: destruction of primary C-afferents with neonatal capsaicin does not affect the number of [3H]clonidine binding sites in mice.

Mice were treated with 50 mg/kg capsaicin on the second day of life, which causes a permanent and selective loss of a large proportion of afferent C-fibers. Two months later the spinal cords were removed and the content of alpha 2-adrenoceptors assayed by radioligand binding with [3H]clonidine. In these tests the content of alpha 2-adrenoceptors was found to be 568 +/- 27 fmol/mg protein in the capsaicin-treated animals and it was virtually the same -596 +/- 9 fmol/mg protein - in control animals. The affinity of [3H]clonidine for the alpha 2-adrenoceptor was not affected by capsaicin (Kd = 3.25 +/- 0.29 nM for capsaicin and 3.23 +/- 0.32 nM for the controls). In functional tests clonidine applied s.c. induced a marked reduction in the pain sensitivity to capsaicin. The results indicate that spinal cord alpha 2-adrenoceptors are located postsynaptically with respect to the primary sensory neurons and potentially that stimulation of these receptors by alpha 2-adrenoceptor agonist mediates inhibition of nociceptive transmission.

Animals↗
PubMed1987

Effects of halothane and other chlorinated hydrocarbons on alpha 2-adrenoceptors in the mouse cortex.

A number of general anaesthetics and organic solvents were tested for their ability to inhibit the binding of 3H-clonidine to alpha 2-adrenoceptors in mouse cerebral cortex membranes. The order of potency of the tested agents was: chloroform greater than halothane greater than trichloroethylene greater than carbon tetrachloride greater than dichloromethane. Of these agents halothane was tested further. When saturation curves of 3H-clonidine were constructed, halothane (25 mmol/l added directly to the assay) was found to induce a proportionally greater inhibition at low 3H-clonidine concentrations than at high. Computer modelling these saturation curves indicated that halothane reduced the apparent affinity of 3H-clonidine; Kd = 4.2 nmol/l in the absence of halothane and Kd = 6.0 nmol/l in its presence. Gassing the cortex membranes with 3% halothane induced a practically identical reduction in the affinity for 3H-clonidine; Kd = 4.6 nmol/l for the control versus Kd = 10.7 nmol/l for halothane. The effects of halothane was compared to that of the non-hydrolyzable GTP analog Gpp(NH)p. Gpp(NH)p in the concentration range 10(-8)-10(-3) mol/l dose-dependently reduced the binding of 1 nmol/l 3H-clonidine, the effect being essentially maximal at 10(-4) mol/l. Computer modelling of saturation curves of 3H-clonidine indicated that 0.1 mmol/l Gpp(NH)p reduced the apparent affinity of 3H-clonidine; Kd = 5.4 nmol/l in the absence of Gpp(NH)p and Kd = 9.3 nmol/l in its presence. In addition Gpp(NH)p caused some reduction in the apparent number of 3H-clonidine binding sites. The effect of halothane on 3H-clonidine binding was tested both in the absence and presence of 0.1 mmol/l 1 Gpp(NH)p. During these conditions halothane was slightly more potent in the presence of Gpp(NH)p (IC50 of halothane = 17 mmol/l) than in its absence (IC50 = 41 mmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Structural and pharmacological differences between codfish and rat brain alpha 1-adrenergic receptors revealed by photoaffinity labeling with 125I-APDQ.

The photoaffinity probe 125I-APDQ has been used to characterize alpha 1-receptor peptides in the cod and rat brains. In the cod brain a major specific peptide of Mr = 68,000 could be covalently labeled by 125I-APDQ as revealed by SDS-PAGE. In the rat brain a specific peptide with Mr = 77,000 was instead labeled. When a number of adrenergic agonists and antagonists were tested for their ability to protect the labeling by 125I-APDQ their potencies were those expected for alpha 1-receptors in both species. The ligand binding peptide in the cod brain also distinguished between stereoisomers of epinephrine as expected for a physiological receptor. However, there was a distinct difference between the cod and rat alpha 1-receptor in that the beta-agonist 1-isoprenaline was equipotent to 1-norepinephrine in the cod whereas it was less potent in the rat. The protecting ability of the tested agents were also matched by their ability to displace the alpha 1-adrenergic ligand 3H-prazosin from alpha 1-receptor binding sites in brain membranes from both species. Thus, the codfish alpha 1-receptor seems to be different from mammalian alpha 1-receptors both structurally and pharmacologically.

Affinity Labels↗
PubMed1986

Guanine nucleotides regulate both agonist and antagonist binding to cod brain alpha 1-adrenoceptors.

The effect of guanine nucleotides on the binding of 3H-prazosin and its displacement by various agonists and antagonists were studied in membranes prepared from the cod brain. When cod brain membranes were maintained in a buffer containing 8 mM Mg2+, GTP (1 mM) was found to increase the specific binding of 3H-prazosin Computer modelling 3H-prazosin saturation curves, suggested that the number of binding sites for 3H-prazosin was increased by 18 +/- 5% by GTP without affecting the affinity for 3H-prazosin (dissociation constant, Kd, 56 +/- 3 pM). Displacement of 3H-prazosin by adrenaline produced shallow displacement curves. Computer modelling these curves suggested that adrenaline bound to two sites - one high and one low affinity site - the Kd's being 0.14 +/- 0.03 (KH) and 7.6 +/- 0.5 microM (KL), respectively. When 1 mM GTP was present the displacement curves were shifted to the right and became steeper. Computer modelling these curves suggested that adrenaline now bound to only one low affinity site with a Kd of 7.6 +/- 0.5 microM. When unlabelled prazosin was used to displace 3H-prazosin the displacement curves were uniphasic and steep, irrespective of whether GTP was present or not. Computer modelling these curves suggested that prazosin bound to only one site with a Kd of 68 +/- 11 pM. In the absence of Mg2+ and in the presence of EDTA (0.8 mM) the displacement curve of adrenaline became steeper and the effect of GTP was almost abolished. During these conditions the ability of GTP to enhance 3H-prazosin binding was also abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1986

Guanylate cyclase activity in normal and neoplastic squamous epithelia from the upper aero-digestive tract.

Cyclic nucleotides (cAMP, cGMP) are suggested to participate in the regulation of cell growth and differentiation. Guanylate cyclase is the enzyme which catalyzes the synthesis of cGMP. The basal guanylate cyclase activity was slightly higher in well-differentiated squamous cell carcinomas than in normal mucosa, but was two-fold higher in papillomas. Poorly differentiated squamous cell carcinoma did not show any increased basal activity. Stimulation with nitroprusside (NP) resulted in a 20% increased activity for normal mucosa and a 30% increase for poorly differentiated carcinomas, whereas enzyme prepared from well-differentiated squamous carcinomas and papillomas showed a two-fold increase.

Carcinoma, Squamous Cell↗
PubMed1985

The effect of pertussis toxin on alpha-2-adrenoceptor-mediated pigment migration in fish melanophores.

The aggregation of melanin-granules within fish pigment cells (melanophores) can be elicited either by electrical stimulation of intrinsic nerves or by the addition of adrenergic agonists. The pigment aggregation seems to be mediated by alpha-2-adrenoceptors. In this investigation we have used various agonists and antagonists (noradrenaline, (+)- and (-)-adrenaline, isoprenaline, yohimbine and prazosin) to further characterize the pigment-aggregating receptor of Labrus ossifagus. All the results obtained support the notion of alpha-2-adrenoceptor-mediated pigment aggregation. The pertussis toxin, islet-activating protein (IAP), is known to inhibit the alpha-2-adrenoceptor-mediated signal transduction in mammals. We have used IAP to investigated whether fish melanophore alpha-2-adrenoceptors are also inhibited by this toxin. We found that IAP inactivated the alpha-2-adrenoceptor-mediated pigment aggregation in a dose-dependent manner. The inhibitory IAP-effect had a remarkably short onset-time in the melanophores (maximal effect was obtained within 10 min of incubation). Interestingly, binding of an agonist (noradrenaline) to the receptors prevented IAP from exerting its inhibitory action, whereas binding of an antagonist (yohimbine) gave no protection against the IAP-inactivation. In conclusion, the pigment-aggregating receptors of melanophores of L. ossifagus are very similar to the mammalian alpha-2-adrenoceptors. It is possible to inactivate the melanophore receptor system with IAP and this inactivation has a remarkably short onset-time. Stimulation of the alpha-2-adrenoceptors prevents IAP from inactivating the receptor system.

Adrenergic alpha-Agonists↗
PubMed1985

Noradrenaline depletion increases noradrenaline-induced antinociception in mice.

Mice were treated with N-2-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride (DSP4), which causes severe noradrenaline (NA) depletions in brain regions and the spinal cord, or vehicle i.p. They were tested 14 days later for antinociception induced by intrathecal injections of different doses of NA. A potentiation of the NA effect upon pain sensitivity was observed, with both an increase in the magnitude and duration of the antinociceptive responses. Upon biochemical analysis of spinal cords, it was found that DSP4-treated mice had a 80% depletion of NA, whereas dopamine and 5-hydroxytryptamine were unaffected. Radioligand binding of [3H]clonidine in membranes prepared from spinal cord, showed no differences in density of alpha 2-adrenoceptors, but the affinity had been increased, probably explaining the supersensitivity.

Animals↗
PubMed1985

Effect of general anaesthetics and organic solvents on alpha 1-adrenoceptors in the myometrium.

The alpha 1 selective radioligand 3H-prazosin was used to assay alpha 1 receptors in membranes prepared from the rabbit myometrium. 3H-Prazosin was found to bind to a single high affinity site in these membranes which was the presumed alpha 1 receptor. A series of general anaesthetics and organic solvents were tested for their ability to inhibit 3H-prazosin binding. The order of potency of the tested agents to inhibit the binding was: chloroform=halothane=trichloroethylene greater than carbon tetrachloride greater than dichloromethane. The depression of 3H-prazosin binding seemed to be induced on the alpha 1 receptor since non-specific radioligand binding was not affected as revealed by a saturation experiment with 3H-prazosin where halothane was used as inhibiting agent. Computer analysis of the latter experiment also showed that halothane depressed mainly the affinity of 3H-prazosin for the alpha 1 receptor. The ability of the general anaesthetics and organic solvents to inhibit contractions elicited by alpha 1 stimulation with phenylephrine in the rabbit uterus was also investigated. In these tests the order of potency for the inhibition of the contractile response was: carbon tetrachloride greater than or equal to halothane=chloroform greater than trichloroethylene greater than dichloromethane. The mechanism of action for alpha 1 receptor and myometrial depression is discussed.

Anesthetics↗
PubMed1985

Effects of retinylacetate on the kinetics of rat liver guanylate cyclase: possible interaction with sulfhydryl groups.

The effect of retinylacetate (RA) was investigated on rat liver guanylate cyclase stimulated by nitroprusside (NP). The stimulated enzyme seemed to adhere to Michaëlis-Menten kinetics with an apparent Km for MnGTP of 0.10 mM and a Vmax of 410 pmol cGMP/min. X mg prot. RA (0.1-1mM) dose-dependently inhibited the enzyme stimulated by NP (0.1-10 mM). In the presence of 1 mM RA the activity was only 10% of the control activity. The inhibitory action of RA seemed to be non-competitive since it depressed Vmax without affecting the Km for MnGTP. In contrast, however, RA was instead found to stimulate the enzyme when low concentrations of NP (0.01-0.1 mM) were used. The concentration-activity curve for NP was bell-shaped showing an optimum at 0.5 mM. The inhibition induced by RA could not be surmounted by increasing the NP concentration indicating that RA did not compete with NP. A bell-shaped activity curve was also seen when the enzyme activity was measured in the presence of increasing Mn2+ concentrations and during these conditions RA also caused inhibition. In the presence of the sulphydryl reductant dithiothreitol (DTT), the NP concentration needed for optimal enzyme activation was about 100-fold less than in the absence of DTT. The maximal enzyme activity was also slightly increased. In the presence of DTT, RA was much less effective to induce inhibition of the stimulated enzyme, than when DDT was absent. (In the presence of DTT 1 mM RA caused only 30% inhibition compared to 90% inhibition in its absence).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Photoaffinity labeling of mammalian alpha 1-adrenergic receptors. Identification of the ligand binding subunit with a high affinity radioiodinated probe.

We have synthesized and characterized a novel high affinity radioiodinated alpha 1-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido - 3 - [125I]iodophenyl) pentanoyl] - 1 - piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (KD = 130 pM) in a reversible and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an alpha 1-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical alpha 1-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at Mr = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the Mr = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the alpha 1-adrenergic receptor.

Affinity Labels↗
PubMed1984

Inhibition of the excitatory non-adrenergic, non-cholinergic neurotransmission in the guinea pig tracheo-bronchial tree mediated by alpha 2-adrenoceptors.

Guinea pig main bronchi were studied in vitro for the presence of motor innervation. Field stimulation of the main bronchi revealed that besides the excitatory cholinergic component a slow and atropine resistant contraction similar to that previously found in the hilus bronchi (Grundström et al. 1981a) was present also at this level of the airways. This slow contraction was blocked by tetrodotoxin whereas it was left unaffected by a number of conventional antagonists (i.e. atropine, guanethidine, propranolol, yohimbine and prazosin) suggesting that it was due to activation of non-adrenergic, non-cholinergic neurones. In the presence of a beta-adrenoceptor blocking drug, noradrenaline inhibited the slow contractions induced by field stimulation in both the main and hilus bronchi. By contrast, contractions elicited by histamine were left unaffected by noradrenaline. In order to characterize the receptor for noradrenaline the effects of alpha1-and alpha2-adrenoceptor blockers were evaluated. The results indicate that in the guinea pig bronchi noradrenaline inhibits non-adrenergic, non-cholinergic neurotransmission by acting on prejunctional alpha 2-adrenoceptors.

Animals↗