Guinea Pig Cerebral Cortical Slices Research Articles

SB-224289--a novel selective (human) 5-HT1B receptor antagonist with negative intrinsic activity.

1. Human 5-HT1B (h5-HT1B) and human 5-HT1D (h5-HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB-224289 (1'-Methyl-5-[[2'-methyl-4'-(5-methyl- 1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro spiro [furo [2,3-f]indole-3,4'-piperidine] oxalate), which has high affinity for h5-HT1B receptors (pK1=8.16+/-0.06) and displays over 75 fold selectivity for the h5-HT1B receptor over all other 5-HT receptors including the h5-HT1D receptor and all other receptors tested thus far. 2. Functional activity of SB-224289 was measured in a [15S]GTPgammaS binding assay on recombinant h5-HT1B and h5-HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB-224289 displayed negative intrinsic activity at both receptors with higher potency at h5-HT1B receptors. SB-224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies. 3. SB-224289 potentiated [3H]5-HT release from electrically stimulated guinea-pig cerebral cortical slices to the same extent as as the non-selective 5-HT1 antagonist methiothepin. SB-224289 also fully reversed the inhibitory effect of exogenously superfused 5-HT on electrically stimulated release. 4. Using SB-224289 as a tool compound, we confirm that in guinea-pig cerebral cortex the terminal 5-HT autoreceptor is of the 5-HT1B subtype.

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors.

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT1B receptor ligand, and of BRL-15572, a preferential h5-HT1D receptor ligand, on the presynaptic h5-HT1B and h5-HT1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT1D heteroreceptors in the human atrium. The brain preparations, preincubated with [3H]serotonin ([3H]5-HT), and the segments of atrial appendages, preincubated with [3H]noradrenaline, were superfused with modified Krebs' solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K+ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2 microM; concentration 154 times higher than its Ki at h5-HT1D receptors) but was antagonized by SB-216641 (0.1 microM; concentration 100 times higher than its Ki at h5-HT1B receptors; apparent pA2 8.45). SB-216641 (0.1 microM) by itself facilitated, whereas BRL-15572 (2 microM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1 microM) also facilitated, and BRL-15572 (2 microM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K+-evoked tritium overflow. This response was unaffected by BRL-15572 (300 nM) but antagonized by SB-216641 (15 nM; drug concentrations 23 and 15 times higher than their Ki at h5-HT1D and h5-HT1B receptors, respectively). Both drugs, given alone, did not modify the K+-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300 nM; 23 times Ki at h5-HT1D receptors) but not by SB-216641 (30 nM; 30 times Ki at h5-HT1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT1B and 5-HT1D receptors in functional studies.

Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells.

1. An [3H]-adenine pre-labelling methodology was employed to assay cyclic AMP generation by adenosine analogues in Chinese hamster ovary (CHO.A2B4) cells, transfected with cDNA which has been proposed to code for the human brain A2B adenosine receptor, and in guinea-pig cerebral cortical slices. 2. Adenosine analogues showing the following rank order of potency in the CHO.A2B4 cells (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.91) > adenosine (5.69) > 2-chloroadenosine (5.27) > N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.06). The purportedly A2A-selective agonist, CGS 21680, failed to elicit a significant stimulation of cyclic AMP generation at concentrations up to 10 microM in CHO.A2B4 cells. In the guinea-pig cerebral cortex, NECA was more potent than APNEA with pD2 values of 5.91 and 4.60, respectively. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity in CHO.A2B4 cells (ca. 10 fold stimulation of cyclic AMP), while, in comparison, maximal responses to adenosine (32% NECA response), 2-chloroadenosine (61%), and APNEA (73%) were reduced. 4. Antagonists of NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value in CHO.A2B4 cells: guinea-pig cerebral cortex): XAC (7.89: 7.46) > CGS 15943 (7.75: 7.33) > DPCPX (7.16: 6.91) > PD 115,199 (6.95: 6.39) > 8FB-PTP (6.52: 6.55) > 3-propylxanthine (4.63: 4.59). 5. We conclude that, using the agents tested, the A2B adenosine receptor cloned from human brain expressed in Chinese hamster ovary cells exhibits an identical pharmacological profile to native A2B receptors in guinea-pig brain.

Presynaptic 5-HT autoreceptors modulate N-methyl- d-aspartate-evoked 5-hydroxytryptamine release in the guinea-pig brain cortex

Guinea-pig cerebral cortical slices preincubated with [ 3H]5-hydroxytryptamine ([ 3H]5-HT) were superfused with Mg 2+-free Krebs' solution, N-Methyl- d-aspartate (NMDA) stimulated tritium overflow in a concentration-dependent manner. The NMDA-evoked overflow was abolished by omission of Ca 2+ or presence of 1.2 mM Mg 2+, but only partly inhibited by tetrodotoxin. The competitive and noncompetitive NMDA receptor antagonists, dl-( E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849) and dizocilpine, respectively, also blocked the stimulatory effect of NMDA. Furthermore, the NMDA-evoked tritium overflow was inhibited by 5-carboxamidotryptamine in a manner susceptible to blockade by methiothepin, which given alone facilitated overflow. This facilitatory effect was increased in the presence of 6-nitroquipazine, a selective 5-HT reuptake inhibitor. It is concluded that the release of 5-HT in the guinea-pig cerebral cortex is stimulated via NMDA receptors, which are in part located on the serotoninergic axon terminals, and that the NMDA-evoked 5-HT release is modulated via inhibitory 5-HT autoreceptors.

Functional characterization of the 5-HT terminal autoreceptor in the guinea-pig brain cortex.

1 In guinea-pig cerebral cortical slices in vitro we have shown that the rank order of potency of 5-hydroxytrptamine (5-HT), 5-carboxamidotryptamine and sumatriptan for inhibition of electrically stimulated [3H]-5-HT release correlates well with published data on their 5-HT1D receptor binding affinities. 2 Both the non-selective 5-HT1D receptor antagonist, methiothepin and the selective 5-HT1D receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl]phenyl]-2'-methyl-4'- (5-methyl-1,2,4-oxadiazole-3-yl) [1,1-biphenyl]4-carboxamide (GR127935) increased stimulated [3H]-5-HT release per se and also attenuated agonist-induced inhibition of [3H]-5-HT release. GR127935 (10 nM-100 nM) produced a pA2 of 9.0 against 5-HT, which is consistent with its 5-HT1D receptor binding affinity. 3 From these findings we conclude that, in guinea-pig cerebral cortex, the 5-HT terminal autoreceptor is of the 5-HT1D receptor subtype. However, three observations suggest the presence of multiple terminal autoreceptors: shallow inhibition curves to the agonists; a shallow Schild slope of GR127935 antagonism and differences in the maximal responses to 5-HT between whole cortex and frontal cortex.

A1 adenosine receptor inhibition of cyclic AMP formation and radioligand binding in the guinea-pig cerebral cortex.

1. A1 adenosine receptors were investigated by radioligand binding and functional studies in slices and particulate preparations from guinea-pig cerebral cortex. 2. Binding of the adenosine receptor antagonist radioligand, 8-cyclopentyl-[3H]-1,3-dipropylxanthine (DPCPX) to guinea-pig cerebral cortical membranes exhibited high density (1410 +/- 241 fmol mg-1 protein) and high affinity (Kd 3.8 +/- 0.3 nM). 3. [3H]-DPCPX binding to guinea-pig cerebral cortical membranes was displaced in a monophasic manner by adenosine receptor antagonists with the rank order of affinity (Ki values, nM): DPCPX (6) < xanthine amine congener (XAC, 153) < PD 115,199 (308). 4. Agonist displacement of [3H]-DPCPX binding was biphasic and exhibited the following rank order at the low affinity site (Ki values): 2-chloro-N6-cyclopentyl-adenosine (CCPA, 513 nM) = N6-R-phenylisopropyladenosine (R-PIA, 526 nM) = N6-cyclopentyladenosine (CPA, 532 nM) < 2-chloroadenosine (2CA, 3.2 microM) = 5'-N-ethylcarboxamidoadenosine (NECA, 4.6 microM) < N6-S-phenylisopropyladenosine (S-PIA, 19.9 microM). 5. In cerebral cortical slices, [3H]-DPCPX binding was displaced by antagonists and agonists in an apparently monophasic manner with the rank order of affinity (Ki values, nM): DPCPX (14) < XAC (45) < R-PIA (266) < PD 115,199 (666) < S-PIA (21000). 6. Cyclic AMP accumulation stimulated by 30 microM forskolin in guinea-pig cerebral cortical slices was inhibited by R-PIA, CCPA and CPA up to 1 microM in a concentration-dependent fashion with IC50 values of 14, 18, and 22 nM, respectively. All three analogues inhibited the forskolin response to a similar extent (82-93% inhibition). NECA, S-PTA and 2CA failed to inhibit the forskolin response, but rather enhanced the accumulation of cyclic AMP at concentrations of 100 nM or greater, presumably through activation of A2b adenosine receptors coupled to stimulation of cyclic AMP accumulation in guinea-pig cerebral cortical slices.7. The inhibition of forskolin-stimulated cyclic AMP accumulation by CPA was antagonized with the rank order of affinity (Ki values, nM): DPCPX (6)<XAC (52)<PD 115,199 (505).8 Xanthine-based antagonists inhibited the adenosine receptor augmentation of histamine-induced phosphoinositide turnover in guinea-pig cerebral cortical slices with the rank order of affinity (Ki, nM):DPCPX (12)=XAC (17)<PD 155,199 (640).9 In summary, we observe a good correlation between antagonist affinity at A1 receptors defined by radioligand binding, inhibition of cyclic AMP generation or augmentation of histamine-evoked phosphoinositide turnover in guinea-pig cerebral cortex.

MRS studies on calcium and zinc in metabolizing cerebral slices exposed to glutamate and NMDA

The19F-NMR calcium indicator 5FBAPTA has been used to measure changes in free intracellular calcium, [Ca2+],, in superfused guinea pig cerebral cortical slices. Increases in [Ca2+], have been observed with depolarization and the combined insult of hypoxia and hypoglycemia. These increases in [Ca2+]i, can be explained by the excitotoxic hypothesis which proposes a central role for the neurotransmitter glutamate and the NMDA receptor in cell death. We, therefore, investigated the effects of glutamate and NMDA directly on [Ca2+]i, in cerebral cortical slices with31P-NMR to monitor the energy state. In the presence of glutamate (±Mg) a new resonance was observed in the19F spectra and was attributed to the Zn-5FBAPTA complex. The zinc peak appears with or just following an increase in [Ca2+]i, and with a drop in PCr. Similar results were obtained on exposing the tissues to NMDA. Zinc has been reported to be enriched in various parts of the brain and have a multiplicity of possible roles. This is the first time zinc has been shown in actively metabolizing tissue. The observation that the zinc resonance does not appear with depolarization or hypoxia and hypoglycemia suggests that the mechanisms of damage in these latter insults are not solely attributable to the release of excitotoxins.

Modulation of cyclic AMP formation by putative metabotropic receptor agonists

1. The effects of various metabotropic glutamate receptor agonists on [3H]-cyclic AMP accumulation and phosphoinositide hydrolysis were investigated in guinea-pig cerebral cortical slices prelabelled with [3H]-adenine or [3H]-inositol. 2. 1-Aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD), L-2-amino-4-phosphonobutanoate (L-AP4) and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited concentration-dependent inhibitions of forskolin-stimulated [3H]-cyclic AMP accumulation, with IC50 values of 2.1 +/- 0.3, 71 +/- 17 and 0.2 +/- 0.1 microM respectively. 3. 1S,3R-ACPD and L-CCG-I increased the cyclic AMP responses to histamine H2 receptor stimulation with EC50 values of 7 +/- 2 microM and 19 +/- 2 microM respectively. 1S,3R-ACPD (EC50 values 17 +/- 2 microM) and L-CCG-I (EC50 value 15 +/- 3 microM) potentiated the cyclic AMP responses to the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM). This potentiating effect of L-CCG-I was reduced in the presence of a protein kinase C inhibitor, and also in the absence of extracellular calcium. In contrast, L-AP4 inhibited the NECA response in a concentration-dependent manner, with an IC50 value of 120 +/- 20 microM. 4. L-AP4 (at concentrations up to 1 mM) failed to stimulate phosphoinositide hydrolysis in guinea-pig cerebral cortical slices, but both 1S,3R-ACPD (EC50 value 35 +/- 6 microM) and L-CCG-I (approximately 160 microM) elicited concentration-dependent stimulations of phosphoinositide turnover. 5. These results confirm the existence of at least two distinct subtypes of metabotropic receptor in guinea-pig cortex. The data also substantiate previous studies indicating the importance of the agonist L-CCG-I as a pharmacological tool for distinguishing metabotropic receptor subtypes.

Subtypes of metabotropic excitatory amino acid receptor distinguished by stereoisomers of the rigid glutamate analogue, 1-aminocyclopentane-1,3-dicar☐ylate

The1 R,3 R- and1 R,3 S-isomers of 1-aminocyclopentane-1,3-dicar☐ylate (ACPD) failed to stimulate phosphoinositide turnover or modify A 2b adenosine receptor-stimulated cyclic AMP accumulation in guinea-pig cerebral cortical slices. In contrast, both1 S,3 R- and1 S,3 S-ACPD elicited concentration-dependent stimulations of phosphoinositide turnover (EC 50 values 35 and 97 μM, respectively) and potentiated A 2b-stimulated cAMP formation (17 and 58 μM, respectively). When forskolin was used to elevate cyclic AMP levels, however, all four isomers elicited concentration-dependent inhibitions of cyclic AMP formation to the same extent (approximately 90% inhibition). For this response the rank order of potencies were (IC 50 values):1 S,3 S- (0.9 μM) > 1S,3 R- (2.1 μM> 1 R,3 R- (237 μM) > 1 R,3 S-ACPD (∼ 1 mM). These data suggest the presence in guinea-pig cerebral cortex of two distinct subtypes of ACPD receptor coupled to phosphoinositide hydrolysis (and the potentiation of A 2b receptor-stimulated cAMP formation) and the inhibition of forskolin-stimulated cAMP accumulation. Furthermore, our results indicate the usefulness of 1 S,3 S-ACPD as a tool to selectively activate one of these subtypes.

Activation of a metabotropic excitatory amino acid receptor potentiates A 2b adenosine receptor-stimulated cyclic AMP accumulation

Incubation of guinea-pig cerebral cortical slices with the excitatory amino acid agonists dl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N- methyl- d-aspartate (NMDA) failed to significantly alter either basal or 5′- N-ethylcarboxamidoadenosine-(NECA-)stimulated cAMP levels. Quisqualate, l-glutamate and dl-1-aminocyclopentane- trans-1,3-dicarboxylate ( t-ACPD) were also without effect on basal levels of cAMP but increased NECA-stimulated levels of cAMP to approximately 190, 220 and 290% of control levels, respectively. Analysis of t-ACPD concentration-response data for the potentiation of NECA-stimulated cAMP elevations and the stimulation of phosphoinositide turnover gave EC 50 values of 51 and 107 μM, respectively. The enhancing effect of t-ACPD on cAMP levels was maintained for up to 40 minutes as was the stimulation of phosphoinositide turnover. We conclude that the cAMP response to A 2b adenosine receptor stimulation is augmented by the rigid glutamate analogue t-ACPD, possibly through the action of products of phosphoinositide turnover.

Excitatory amino acid-induced phosphoinositide turnover in guinea pig cerebral cortical slices: selective enhancement by spermine of the response to DL-1-aminocyclopentane-trans-1,3-dicarboxylate.

In the presence of 1 mM spermine, accumulations of 3H labelled inositol phosphates elicited by quisqualate (100 microM) and 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD, 300 microM) were significantly enhanced by 21 and 26%, respectively, without a significant alteration in the accumulation elicited by L-glutamate (10 mM) or DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionate (10 microM). Analysis of concentration-response data indicated that the presence of spermine led to an increase in the maximal response to t-ACPD without altering the EC50 value. The stimulatory effect of spermine on the accumulation of t-ACPD-elicited 3H-inositol phosphates was not reversed by ifenprodil or diethylenetriamine (putative polyamine site antagonists), by agents that activate or inhibit protein kinase C, or by calcium channel blockade, but was abolished in the presence of elevated extracellular calcium ion concentration. We conclude that spermine enhances the phosphoinositide turnover in guinea pig cerebral cortical slices elicited by the "metabotropic" excitatory amino acid receptor. The site through which the action of spermine is mediated remains to be defined, but it is apparently distinct from that suggested to modulate N-methyl-D-aspartate receptor activity.

Do polyamines regulate the NMDA inhibition of muscarinic receptor-induced phosphoinositide turnover in guinea pig brain?

The N- methyl- d-aspartate (NMDA) receptor-induced inhibition of muscarinic receptor-stimulated phosphoinositide turnover in guinea pig cerebral cortical slices was investigated to determine whether polyamines regulate the function of NMDA in this system. None of the polyamines tested produced significant stimulation of phosphoinositide turnover. Neither spermine nor the substituted polyamine philanthotoxin-343 altered the inhibitory effect of NMDA. The simpler substituted polyamines N-(4-hydroxyphenylpropanoyl)-spermine and N-(4-hydroxyphenylacetyl)-spermine, however, antagonised the NMDA receptor inhibition of the muscarinic response with low affinity ( K i ≧ 100 μ M ). Diethylenetriamine, a purported polyamine antagonist of the NMDA receptor, was without effect on the NMDA inhibitory response, as was ifenprodil, a structurally unrelated compound with reported antagonist capacity. In summary, therefore, we fail to observe polyamine regulation of the NMDA receptor mediating an inhibition of the muscarinic phosphoinositide turnover response in guinea pig cerebral cortical slices.

Histamine receptors and interactions between second messenger transduction systems.

Histamine H1- and H2-receptors have been classically associated with two distinct intracellular second messenger systems. H2-receptors are coupled via a Gs regulatory protein to adenylate cyclase and stimulate cyclic AMP formation, while H1-receptors mediate many of their effects via the products of inositol phospholipid hydrolysis. It is becoming clear, however, that there is a substantial 'cross-talk' between these intracellular second messenger systems in a number of tissues. In guinea-pig cerebral cortical slices, H1-receptor stimulation can augment the direct cyclic AMP responses to adenosine or H2-receptor stimulation via a mechanism which appears to involve both products of inositol phospholipid hydrolysis, inositol triphosphate and diacylglycerol. In contrast, a reverse interaction can occur in bovine tracheal smooth muscle and agents which raise intracellular cyclic AMP levels can inhibit H1-receptor-mediated inositol phospholipid hydrolysis. In addition to these interactions between cyclic AMP and phosphoinositide signalling systems, the H1-receptor-mediated inositol phospholipid response in mammalian cerebral cortex can be regulated (both positively and negatively) via mechanisms which do not involve changes in cyclic AMP levels.

Is the Adenosine Receptor Modulation of Histamine‐Induced Accumulation of Inositol Phosphates in Cerebral Cortical Slices Mediated by Effects on Calcium Ion Fluxes?

In this report, we show that under conditions designed to provide an initially uniform incorporation of [3H]inositol into mouse and guinea pig cerebral cortical slices prior to agonist stimulation, the accumulation of 3H-inositol phosphates (3H-InsPx, x = 1-4) induced by histamine in mouse and guinea pig cerebral cortical slices increased in a quasilinear manner with increasing added calcium. Raising the ambient calcium ion concentration failed to reduce the adenosine receptor-mediated inhibition of the histamine-induced 3H-InsPx response in mouse cerebral cortical slices. Similarly, the potentiation of the histamine response by adenosine receptor activation in guinea pig cerebral cortical slices was unaffected by lowering the added calcium ion concentration. The presence of the calcium ionophore A23187 (33 microM) produced 3H-InsPx responses in both mouse and guinea pig cerebral cortical slices, which were not affected by the presence of the stable adenosine analogue 2-chloroadenosine. A23187 also potentiated the accumulation of 3HInsPx induced by histamine in both species. Both the inhibitory and potentiatory modulations of the histamine response by 2-chloroadenosine in mouse and guinea pig, respectively, were still apparent in the presence of A23187. These results indicate that the histamine-induced 3H-InsPx accumulations in both mouse and guinea pig cerebral cortical slices are sensitive to variations in calcium ion concentrations. However, the adenosine receptor modulations of the histamine responses are relatively insensitive to fluctuations in either extra- or intracellular calcium ion concentrations, and thus cannot be mediated by effects on calcium ion movements.

Excitatory Amino Acid‐Induced Formation of Inositol Phosphates in Guinea‐Pig Cerebral Cortical Slices: Involvement of Ionotropic or Metabotropic Receptors?

In cerebral cortical slices from the guinea-pig, quinoxalinedione derivatives antagonised the generation of 3H-inositol phosphates evoked by the excitatory amino acids quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid but were without effect on the trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid and L-glutamate responses. Omission of calcium from the medium reduced the accumulation of 3H-inositol phosphates induced by incubation with trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (incubation for 45 min) by greater than 50%, whereas the responses to L-glutamate and the two other amino acid analogues were reduced by approximately 20%. Generation of inositol 1,4,5-trisphosphate over a 30-s period by treatment with quisqualate, trans-DL-1-amino-1,3-cyclopentane-dicarboxylic acid, KCl, and carbachol was abolished in the presence of nominally calcium-free medium. L-Glutamate induced a large, rapid increase in inositol 1,4,5-trisphosphate mass (more than three-fold), which was, however, unaffected by omission of calcium from the medium. These results indicate that of the excitatory amino acids tested, only L-glutamate may be classed as a metabotropic receptor agonist in guinea-pig cerebral cortical slices with respect to generation of inositol phosphates. The other agents appear to stimulate accumulation of inositol phosphates, at least in part through some mechanism requiring the presence of extracellular Ca2+, presumably Ca2+ entry.

Inositol phosphates accumulation in guinea pig cerebral cortical slices: effects of protein kinase C activators and an inhibitor

Inositol phosphates accumulation in guinea pig cerebral cortical slices: effects of protein kinase C activators and an inhibitor

Effect of Membrane Depolarization by High K+ on Carbachol-Stimulated Phosphoinositides Hydrolysis in Guinea Pig Cerebral Cortical Slices

Stimulation of phosphoinositide hydrolysis by carbachol was studied in slices of guinea pig cerebral cortex under normal conditions (4.7 mM K+) and depolarization conditions with high K+ (42 mM K+). Slices were labeled with [myo-3H]-inositol, and the effects of carbachol and high K+ on the formation of inositol-bisphosphates (IP2) and inositol-trisphosphates (IP3) were determined. Carbachol (10 mM) caused only 140% stimulation of the formations of IP2 and IP3 over the control value in normal Krebs Ringer Buffer (KRB), but about 200% stimulation in high K+ medium. Dose-response curves for the effect of carbachol on the formations of IP2 and IP3 showed that high K+ medium selectively decreased the ED50 value of carbachol for IP2 formation about 3-fold. A Ca++ channel blocker, verapamil, inhibited the synergistic effect of carbachol and high K+ on IP2 formation, and a decrease in extracellular Ca++ also inhibited IP2 formation induced by high K+, but these treatments had little, if any, effect on IP3 formation. The possibility that IP2 may be directly generated by hydrolysis of phosphatidylinositol 4-monophosphate (PIP) as well as from hydrolysis of IP3 was discussed.

Differences in the adenosine receptors modulating inositol phosphates and cyclic AMP accumulation in mammalian cerebral cortex

1. 2-Chloroadenosine stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation and potentiated (guinea-pig) or inhibited (mouse) the histamine H1-receptor-stimulated hydrolysis of inositol phospholipids in slices of guinea-pig and mouse cerebral cortex. 2. Two xanthine-based adenosine receptor antagonists were identified which were one order of magnitude more potent at the adenosine receptor mediating augmentation of the histamine-stimulated inositol phospholipid hydrolysis than at the receptor linked to cyclic AMP formation in guinea-pig cerebral cortical slices. 3. These compounds, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 7-benzyl-3-(2-methylpropyl)xanthine (BMPX) retained their selectivity under near-identical incubation conditions in both assays. 4. These compounds also showed similar affinities and selectivity for the adenosine receptor mediating inhibition of histamine-stimulated inositol phospholipid hydrolysis in mouse cerebral cortical slices. 5. Inclusion of agents which give rise to, or mimic, high levels of cyclic AMP (forskolin (1 microM), 8-bromo-cyclic AMP (1 mM) and vasoactive intestinal polypeptide (1 microM] in the incubation medium failed to mimic the action of 2-chloroadenosine on inositol phospholipid turnover in cerebral cortical slices from either species. 6. These data suggest that the adenosine receptor modulating the hydrolysis of inositol phospholipid in mouse and guinea-pig cerebral cortical slices is different from the adenosine receptor linked to cyclic AMP formation, and, furthermore, that the modulation of inositol phospholipid metabolism in either species is not mediated via alterations in cyclic AMP levels.

Temporal changes in the calcium‐dependence of the histamine H1‐receptor‐stimulation of cyclic AMP accumulation in guinea‐pig cerebral cortex

1. 2-Chloroadenosine (2CA) causes a maintained rise in adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of guinea-pig cerebral cortical slices which is augmented by addition of histamine. We have investigated the temporal profile of the sensitivity of this response to calcium. 2. Rapid removal of extracellular calcium with EGTA (5 mM) at 2CA (30 microM)-induced steady state caused a slight increase in the cyclic AMP response to 2CA alone and completely abolished the augmentation produced by histamine (0.1 mM) added 20 min later. When EGTA was added only 2 min before histamine, the augmentation was reduced by 72%. 3. The calcium sensitivity of the histamine response was also indicated in studies in which EGTA was added 1 or 3 min after histamine at 2CA-induced steady state. Following addition of EGTA at either of these times, the augmentation was not maintained. 4. When calcium was rapidly removed with EGTA once a steady state level of cyclic AMP had been achieved with histamine, the augmentation response was maintained. This was despite the fact that EGTA had a similar effect on both extracellular free calcium and tissue calcium content when it was applied before or after histamine. 5. The 2CA response was augmented by phorbol esters (which mimic the actions of diacylglycerol) in a calcium-independent manner. 6. These results suggest that calcium is important for the initiation and early stages of the histamine-induced augmentation response. The apparent lack of calcium sensitivity of the response at later stages could mean that calcium is not involved in the maintenance of the response or that the intracellular machinery involved in the augmentation process becomes more sensitive to calcium as the response progresses, such that it becomes able to operate at a much lower level of intracellular calcium. A possible role for diacylglycerol in the maintenance of the response is discussed.

P-342 - High K+ enhances carbachol-stimulated PI turnover in guinea pig cerebral cortical slices

P-342 - High K+ enhances carbachol-stimulated PI turnover in guinea pig cerebral cortical slices