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K Starke

Publications and source records attributed to K Starke.

At least 37 records · Page 2Linked to original sources
PubMed2000

Electrically evoked release of [(3)H]noradrenaline from mouse cultured sympathetic neurons: release-modulating heteroreceptors.

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting alpha(2)-autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [(3)H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M(2) muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB(1) receptors. Prostaglandin E(2), sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E(2), and somatostatin were abolished (at the highest concentration of WIN 55, 212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the beta(2)-adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.

Action Potentials↗
PubMed1999

A search for presynaptic imidazoline receptors at rabbit and rat noradrenergic neurones in the absence of alpha 2-autoinhibition.

Presynaptic, release-inhibiting imidazoline receptors have hitherto been detected mainly under conditions of ongoing alpha 2-autoinhibition. We tried to find them under alpha2-autoinhibition-free conditions, in the majority of experiments in the rabbit pulmonary artery but in a few experiments also in rabbit atria, rabbit brain cortex and rat brain cortex. Tissue segments were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Ten compounds, some thought to inhibit noradrenaline release through activation of presynaptic imidazoline receptors, some thought to act through alpha 2-adrenoceptors, were tested. Rauwolscine and 6-chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine (SKF 86466) were used as antagonists. In rabbit pulmonary artery segments stimulated by trains of 20 pulses/50 Hz (alpha 2-autoinhibition-free conditions), all ten "agonists" [medetomidine, aganodine, 4-chloro-2-guanidyl-isoindoline (BDF 7579), 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143), 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), alpha-methylnoradrenaline, clonidine, moxonidine, cirazoline and idazoxan] reduced the stimulation-evoked overflow of tritium, with potency decreasing in that order and with maximal inhibition by 59% (idazoxan) to 95% (moxonidine). Rauwolscine competitively antagonized the effects of all ten "agonists" with similar potency, the pKd-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pKd 7.6). Cirazoline was also studied in the other three tissues. In rabbit atrial segments stimulated by 20 pulses/50 Hz and rabbit brain cortex slices stimulated by 4 pulses/100 Hz (both autoinhibition-free), cirazoline reduced the evoked overflow of tritium with concentration-inhibition curves similar to the curve in the pulmonary artery. In the brain cortex, the pKd-value of rauwolscine against cirazoline was 7.7 (pulmonary artery: 7.6). In rat brain cortex slices stimulated by 3 pulses/100 Hz (autoinhibition-free), cirazoline failed to change the evoked overflow of tritium but antagonized the inhibitory effect of UK 14304, pKd of cirazoline against UK 14304 6.9. In rat brain cortex slices stimulated by trains of 36 pulses/3 Hz, finally (marked alpha 2-autoinhibition), cirazoline increased the evoked overflow of tritium. In the rabbit pulmonary artery, rauwolscine and SKF 86466 acted with the same potency against typical presynaptic imidazoline receptor agonists (such as clonidine) and typical alpha 2-adrenoceptor agonists (such as moxonidine). Hence, presynaptic imidazoline receptors were not detectable, in a tissue that is prototypical for presynaptic imidazoline receptors, in the absence of alpha 2-autoinhibition, i.e. under conditions usually thought to be optimal for presynaptic receptor characterization. The pKd-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the alpha 2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an alpha 2(A)-autoreceptor agonist in rabbit tissues but as an alpha 2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) alpha 2A-adrenoceptors than the orthologous (rodent) alpha 2D-adrenoceptors.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed1999

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens.

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by alpha,beta-MeATP (3 microM), with IC50-values ranging from 1.1 to 100 microM. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8'-[Carbonylbis(imino-3, 1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate ) (NF023), suramin, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34-43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-4-amino-5-hydroxy-naphtha lene-1,3-disulphonate (NH01) and Evans blue even enhanced the response to ATP. For antagonists causing full or partial inhibition, the IC50-values against ATP were close to those against alpha,beta-MeATP. All antagonists attenuated the removal of ATP, with IC25%-values ranging from 0.8 microM to >320 microM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X1). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones.

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 microM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 microM) and cibacron blue 3GA (3 microM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 microM; by 47%), by the N-type calcium channel blocker omega-conotoxin GVIA (100 nM; by 35%), and by the alpha2-adrenoceptor agonist UK-14,304 (1 microM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by omega-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 microM) caused a marked increase in intra-axonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and omega-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 microM) induced a large depolarisation and action potential firing. UDP (100 microM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and - probably - the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by alpha2-adrenoceptors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to alpha2-adrenoceptor modulation.

Action Potentials↗
PubMed1999

A contraction-mediating receptor for UTP, presumably P2Y2, in rat vas deferens.

The possible existence of a contraction-mediating P2-receptor for uracil nucleotides was investigated in the rat vas deferens. In order to minimize breakdown of nucleotides, Evans blue was used as an inhibitor of ectonucleotidases. UTP was degraded by rat vas deferens tissue, and the degradation was inhibited by Evans blue (100 microM). In the absence of other drugs, UTP and UDP elicited marginal contractions. Evans blue (100 microM) greatly enhanced contractions elicited by the uracil nucleotides. When the medium contained alpha,beta-MeATP (100 microM) in addition to Evans blue in order to desensitize contraction-mediating P2X1-receptors, responses to UTP and UDP were not changed; in contrast, responses to alpha,beta-MeATP were virtually abolished and contractions elicited by ATP and ADP were greatly reduced; EC50 values were 122 microM for UTP and 58 microM for ATP under these conditions. The P2-receptor antagonist suramin attenuated contractions elicited by UTP (320 microM) and alpha,beta-MeATP (32 microM) in the presence of Evans blue; pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) also reduced responses to alpha,beta-MeATP but, at up to 100 microM, did not alter contractions elicited by UTP. Incubation of vasa deferentia in nominally calcium-free medium almost abolished the response to alpha,beta-MeATP (32 microM), while a major part of the contraction elicited by UTP (320 microM) was preserved. In the presence of Evans blue and alpha,beta-MeATP, prior addition of UDP (3200 microM) or ATP (320 microM), without washout, markedly reduced the response to UTP (320 microM); UTP and ATP also reduced the response to UDP; in contrast, prior addition of UTP or UDP did not alter the contraction to ATP. The results demonstrate the existence of a contraction-mediating, uracil nucleotide-sensitive P2Y-receptor in rat vas deferens, distinct from the P2X1-receptor. Pharmacological analysis indicates that it is P2Y2.

Adenosine Diphosphate↗
PubMed1999

Occurrence, pharmacology and function of presynaptic alpha2-autoreceptors in alpha2A/D-adrenoceptor-deficient mice.

The occurrence, pharmacological properties and function of alpha2-autoreceptors were studied in hippocampal slices, occipito-parietal cortex slices, segments of heart atria and segments of the vas deferens of wildtype (WT) mice and mice in which the alpha2 A/D-adrenoceptor gene had been disrupted (alpha2 A/D(KO)). Tissues were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation periods for brain slices consisted either of 1 pulse or of 2-64 pulses delivered at 1-s intervals; stimulation periods for peripheral tissues consisted either of 1 POP (pseudo-one-pulse; brief burst of 20 pulses/50 Hz) or of 2-4 POPs delivered at 1-s intervals. Single pulses or POPs were used to study the effect of medetomidine and its interaction with antagonists. One or more pulses or POPs per stimulation period were used to study alpha2-autoinhibition. Medetomidine decreased the evoked overflow of tritium in WT tissues. In alpha2 A/D(KO) tissues, the inhibition was slightly (peripheral tissues) or greatly (brain slices) attenuated but not abolished. Phentolamine, rauwolscine, spiroxatrine, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tolazoline and prazosin antagonized the effect of medetomidine in all tissues. Their pKd values against medetomidine were compared with pKd values at prototypical alpha2 binding sites by means of a correlation analysis. For WT brain and atrial autoreceptors, the correlations indicated an alpha2D pharmacology, whereas for WT vas deferens autoreceptors they favoured an alpha2B pharmacology. In the KO tissues, any correlation with alpha2D was lost, and the non-alpha2 A/D-autoreceptors displayed alpha2B or alpha2C pharmacology. When 2 or more pulses or POPs were applied to WT tissues per stimulation period, the pulse number-overflow curve (POP number-overflow curve) was flat, indicating that overflow elicited by p pulses (POPs) was much smaller than p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused a pulse (POP) number-dependent and, at high pulse (POP) numbers, large increase in evoked tritium overflow. In alpha2 A/D(KO) tissues, the pulse (POP) number-overflow curve was much steeper, indicating that overflow elicited by p pulses (POPs) was closer to p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused no (atria) or only a small increase in overflow, and did so in brain slices only at high pulse numbers (16 and 64). In conclusion, the predominant alpha2D pharmacology of the autoreceptors in WT tissues supports the idea that the main mammalian presynaptic alpha2-autoreceptors belong to the alpha2 A/D subtype. However, alpha2 A/D-deficient animals also possess autoreceptors. As expected, these non-alpha2 A/D-autoreceptors display alpha2B or alpha2C pharmacology. In WT animals, alpha2B- or alpha2C-autoreceptors or both may coexist with alpha2 A/D-autoreceptors, at least in peripheral tissues. Little autoinhibition by released noradrenaline in trains of pulses remains when the alpha2 A/D-adrenoceptor is lacking, again in accord with a predominance of alpha2 A/D-autoreceptors.

Adrenergic alpha-Agonists↗
PubMed1999

A study of the mechanism of the release of ATP from rat cortical astroglial cells evoked by activation of glutamate receptors.

Glutamate and the selective agonists at ionotropic glutamate receptors N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate release ATP from superfused primary cultures of rat cortical astrocytes. The mechanism of this release was investigated. The release of ATP elicited by N-methyl-D-aspartate and kainate was abolished or greatly reduced in the absence of external calcium as well as in the presence of cadmium (1 mM) and nicardipine (10 microM). The release of ATP elicited by AMPA, in contrast, was not changed by these interventions. The calcium ionophore ionomycin (5 microM) released ATP in the presence but not in the absence of external calcium. No release was obtained with alpha-latrotoxin. Of several compounds tested as potential blockers of ATP transporters or channels only glibenclamide (100 microM) and diphenylamine-2-carboxylate (500 microM), which block the cystic fibrosis transmembrane conductance regulator, caused any change: both reduced the effect of AMPA without changing the effects of N-methyl-D-aspartate and (only glibenclamide tested) kainate. Lithium (1 mM) abolished the release of ATP evoked by glutamate and AMPA and significantly reduced the release evoked by N-methyl-D-aspartate and kainate. The three glutamate receptor agonists did not increase the release of lactate dehydrogenase. The results confirm the previous observation that activation of N-methyl-D-aspartate, AMPA and kainate receptors induces release of ATP from astrocytes in culture. Two different mechanisms seem to be involved. The N-methyl-D-aspartate- and kainate-induced release of ATP requires an influx of calcium, is not due to neuron-like exocytosis, is not mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is reduced (by an unknown mechanism) but not abolished by lithium. The AMPA-induced release does not require extracellular calcium, may be mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is abolished (by an unknown mechanism) by lithium. The ability of astrocytes to both release ATP and respond to ATP suggests that ATP may act as an autocrine or paracrine messenger between these glial cells.

Adenosine Triphosphate↗
PubMed1999

Prejunctional angiotensin receptors involved in the facilitation of noradrenaline release in mouse tissues.

The effect of angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) on the electrically induced release of noradrenaline was studied in preparations of mouse atria, spleen, hippocampus, occipito-parietal cortex and hypothalamus preincubated with [3H]-noradrenaline. The prejunctional angiotensin receptor type was investigated using the non-selective receptor antagonist saralasin (AT1/AT2) and the AT1 and AT2 selective receptor antagonists losartan and PD 123319, respectively. In atrial and splenic preparations, angiotensin II (0.01 nM-0.1 microM) and angiotensin III (0.01 and 0.1 nM-1 microM) increased the stimulation-induced overflow of tritium in a concentration-dependent manner. Angiotensin IV, only at high concentrations (1 and 10 pM), enhanced tritium overflow in the atria, while angiotensin-(1-7) (0.1 nM-10 microM) was without effect in both preparations. In preparations of hippocampus, occipito-parietal cortex and hypothalamus, none of the angiotensin peptides altered the evoked overflow of tritium. In atrial and splenic preparations, saralasin (0.1 microM) and losartan (0.1 and 1 microM), but not PD 123319 (0.1 microM), shifted the concentration-response curves of angiotensin II and angiotensin III to the right. In conclusion, in mouse atria and spleen, angiotensin II and angiotensin III facilitate the action potential induced release of noradrenaline via a prejunctional AT1 receptor. Only high concentrations of angiotensin IV are effective in the atria and angiotensin-(1-7) is without effect in both preparations. In mouse brain areas, angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) do not modulate the release of noradrenaline.

Angiotensin II↗
PubMed1999

Mouse postganglionic sympathetic neurons: primary culturing and noradrenaline release.

Basic properties of noradrenaline release were studied in primary cultures of thoracolumbar postganglionic sympathetic neurons taken from 1-3-day-old NMRI mice. After 7 days in vitro, the cultures were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Conventional trains of pulses (for example, 36 pulses at 3 Hz) as well as single pulses and brief high-frequency trains (for example, four pulses at 100 Hz) elicited a well-measurable overflow of tritium, which was abolished by 0.3 microM tetrodotoxin or omission of Ca2+, but not changed by 1 microM rauwolscine. In trains of one, two, four, six, eight, or 10 pulses at 3 Hz, the evoked overflow of tritium remained constant from pulse to pulse at 1.3 mM Ca2+, but declined slightly at 2.5 mM Ca2+. Tetraethylammonium at 10 mM selectively increased the overflow elicited by small pulse numbers and especially by a single pulse. In trains of 10 pulses delivered at 0.3, 1, 3, 10, 30, or 100 Hz, the evoked overflow of tritium increased from 0.3 to 30 Hz and then declined at 100 Hz. This relationship was particularly pronounced at low Ca2+ concentrations (for example, 0.3 mM). Tetraethylammonium at 10 mM selectively increased the overflow elicited by low frequencies of stimulation. It is concluded that primary cultures of mouse postganglionic sympathetic neurons can be used to investigate release of [3H]noradrenaline. The release is well measurable, even upon a single electrical pulse. It agrees with release in intact sympathetically innervated tissues in a number of fundamental properties, including the pulse number and frequency dependence. The preparation may be of special interest in conjunction with genetic manipulations in the donor animals.

Animals↗
PubMed1999

Noradrenaline release from cultured mouse postganglionic sympathetic neurons: autoreceptor-mediated modulation.

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.

Adenosine↗
PubMed1999

Abnormal regulation of the sympathetic nervous system in alpha2A-adrenergic receptor knockout mice.

alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.

Animals↗
PubMed1998

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

Adenosine↗
PubMed1998

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

Animals↗
PubMed1998

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2.

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

Adenosine Triphosphate↗
PubMed1998

P2-receptor-mediated inhibition of noradrenaline release in the rat pancreas.

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM) and yohimbine (1 microM), and stimulated electrically, in most experiments by 60 pulses/l Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA; EC50 32 nM), the non-subtype-selective adenosine receptor agonists adenosine (EC50 15 microM) and 5'-N-ethylcarboxamidoadenosine (NECA; EC50 135 nM), and the nucleotides ATP (EC50 13 microM), adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; EC50 19 microM) and adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS; EC50 16 microM) decreased the evoked overflow of tritium. The adenosine A2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5 '-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A -antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10 nM; pKd 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30 microM). Combined administration of a high concentration of DPCPX (1 microM) and 8-phenyltheophylline (10 microM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPbetaS were shifted to the right by both DPCPX (10 nM; pKd 8.7 and 8.9, respectively) and cibacron blue 3GA (30 microM; pKd 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10 nM) and cibacron blue 3GA (30 microM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1 microM) and 8-phenyltheophylline (10 microM), ATP and ADPbetaS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30 microM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)- 1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A1-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia.

Animals↗
PubMed1998

Inhibition of GABAergic inhibitory postsynaptic currents by cannabinoids in rat corpus striatum.

Electrophysiological consequences of activation of cannabinoid receptors have been mostly investigated on neuronal cell lines and on cells transfected with cannabinoid receptors. The aim of the present experiments was to study cannabinoid effects on identified neurons in situ. Electrically-evoked postsynaptic currents and voltage-dependent calcium currents were investigated in the principal neurons of the corpus striatum, the medium spiny neurons, with the patch-clamp method for brain slices. These neurons were chosen because they produce messenger RNA for cannabinoid receptors and because the density of cannabinoid binding sites in the striatum is high. Activation of muscarinic receptors by carbachol (10(-5) M) reduced inhibitory postsynaptic current amplitude by 67%. The synthetic cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4- benzoxazin-yl]-(1-naphtalenyl)methanone (WIN55212-2; 10(-8) to 10(-5) M) dose-dependently reduced striatal inhibitory postsynaptic currents; the maximum effect, inhibition by 52%, was observed at 10(-6) M. Another cannabinoid agonist, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydr oxypropyl)cyclohexanol (CP55940; 10(-6) M), also reduced inhibitory postsynaptic currents, by 50%. The CB1 cannnabinoid receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)4-methyl-3-pyra zolecarboxamide (SR141716A; 10(-6) M) had no effect when given alone but abolished the effect of WIN55212-2 (10(-6) M). WIN55212-2 (10(-6) M) did not change the current evoked by the GABA(A)-receptor agonist muscimol (10(-6) M). Activation of muscarinic receptors by carbachol (10(-5) M) inhibited voltage-dependent calcium currents by 21%, but the cannabinoid receptor agonist WIN55212-2 (10(-6) M) was without effect. The results show that activation of CB1 cannabinoid receptors reduces GABAergic inhibitory postsynaptic currents in medium spiny neurons of the corpus striatum: the likely mechanism is presynaptic inhibition of GABA release from terminals of recurrent axons of the medium spiny neurons themselves.

Animals↗