Angiotensin receptor-like immunoreactivity in adult brain white matter astrocytes and oligodendrocytes.

Most of the physiological effects of brain angiotensins are currently believed to be mediated by angiotensin receptors located principally on neurons. However, numerous studies in vitro have demonstrated the presence of functional angiotensin receptors on brain astrocytes, raising the possibility that glial cells may also participate in mediating the effects of the central renin-angiotensin system. Nevertheless, it is uncertain whether these cells in situ express angiotensin receptors, raising questions about the physiological significance of results observed in cell cultures. We have examined the distribution of angiotensin receptor-like immunoreactivity in glial cells in white matter tracts in the adult CNS, using a panel of antisera to the AT1 and AT2 angiotensin receptors. Antiserum preadsorption and/or Western blot demonstrated the specificity of the antisera in brain tissue. In immunohistochemical experiments, the AT1 antisera selectively labeled AT1-expressing neurons in the piriform cortex, whereas the AT2 antiserum stained cells in the trigeminal motor nucleus, these being nuclei known to express AT1 and AT2 receptors, respectively. Using double-label immunohistochemistry, we observed AT1- and AT2-immunoreactive astrocytes and oligodendrocytes in white matter tracts, which include the rat cerebellar white matter, periventricular white matter, and optic nerve, in addition to the bovine corpus callosum and human subcortical white matter. In contrast, astrocytes in the gray matter region of the cerebral cortex were not found to be angiotensin receptor-like immunoreactive. These results demonstrate the presence of AT1 and/or AT2 angiotensin receptor-like immunoreactivity in brain white matter macroglial cells in situ and support the idea that glial cells may play a more important role in the central renin-angiotensin system than previously thought.

KA1-like kainate receptor subunit immunoreactivity in neurons and glia using a novel anti-peptide antibody.

Functional kainate receptors can be formed by various combinations of subunits with low (GluR5, GluR6 and GluR7) or high affinity (KA1 and KA2) for kainate. The precise contribution of each subunit to native receptors, as well as their distribution within the central nervous system (CNS) is still unclear. Here, we describe the presence of KA1-like immunoreactivity in both neurons and glial cells of the CNS, using a newly developed antiserum to a specific carboxy terminus epitope of the KA1 subunit. Intense immunoreactivity was observed in the CA3 area of the rat hippocampus. Electron microscopy revealed that immunostaining was present in dendritic structures postsynaptic to commissural-associational fibers, rather than in those contacted by mossy fiber terminals. We also observed immunostaining of CA1 pyramidal cell apical dendrites. In the cerebral cortex, KA1-like immunostaining was observed in many pyramidal neuron somata, mainly in layer V, and along their apical dendrites. A subset of gamma-amino-butyric acidic cells were also intensely stained. In the cerebellum, the antiserum selectively stained Purkinje cell somata and their dendrites as well as Bergmann glial processes. Other types of macroglia were also labeled by the KA1 antiserum. Thus, optic nerve oligodendrocytes both in vitro and in situ and cultured astrocytes were densely stained. Our results indicate that KA1-type subunits are more widely distributed throughout the CNS than previously thought. This newly developed antiserum may help to clarify the properties of kainate receptors containing KA1 or KA1-type subunits within the normal and pathological brain.

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.

On how altered glutamate homeostasis may contribute to demyelinating diseases of the CNS.

Glial cells communicate reciprocally with neurons in multiple ways, both in synaptic and non-synaptic regions of the central nervous system. In the latter, neuron to glial and glial to glial signals can be mediated by neurotransmitters. Here, we review the presence and some of the functional properties of glutamate transporters and receptors in oligodendrocytes. In addition, we present data illustrating that alterations in glutamate homeostasis can be excitotoxic to oligodendroglia and that the tissue lesions caused by overactivation of glutamate receptors resemble those observed in demyelinating diseases such as multiple sclerosis. Overall, this information indicates that aberrant glutamate signaling may contribute to the development of some white matter pathologies.

Cloning and expression of a P2y purinoceptor from the adult bovine corpus callosum.

We have isolated an ATP receptor clone by screening a bovine corpus callosum cDNA library. The clone includes one open reading frame encoding for a protein of 373 amino acid residues (42 kDa) which belongs to the G-protein-coupled receptor superfamily. In Xenopus oocytes, this clone expressed an ATP receptor that triggered an oscillatory current in response to ATP (EC50 approximately 20 microM). This current may have resulted from the activation of phospholipase C, the formation of inositol trisphosphate, and the release of Ca2+, which then opens Cl- channels. The order of potency for ATP receptor agonists was 2-MeSATP approximately ATP >> alpha, beta-MeATP > adenosine, and UTP was ineffective, a pharmacological profile consistent with that of a P2y purinoceptor. Northern blot analysis of mRNAs from various bovine brain tissues showed that the gene is expressed in the cerebellum, medulla, corpus callosum, hippocampus, superior colliculus, frontal cortex, and retina. In situ RT-PCR showed transcripts of the gene in many glial cells and endothelial cells of the corpus callosum. The cloned receptor may play an important role in neuron-glial signaling under normal and pathological conditions.

Activation by P2X7 agonists of two phospholipases A2 (PLA2) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA2 with kallikrein secretion.

Isolated ductal cells of rat submandibular gland phospholipid pools were labeled with [3H]arachidonic acid (AA). The tracer was incorporated preferentially to phosphatidylcholine (46% of the lipidic fraction). Extracellular ATP induced the release of [3H]AA to the extracellular medium in a time- and dose-dependent manner (EC50 = 220 microM). Among other agents tested, only 2', 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was able to mimic the effect of ATP (EC50 = 15 microM), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mM ATP and 100 microM Bz-ATP; the response was also blocked by the addition of Mg2+ or Ni2+. Expression of P2X7 receptor mRNA in these cells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A2 inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific inhibitor), arachidonyl trifluoromethylketone (AACOCF3, an inhibitor of the calcium-dependent cytosolic PLA2 (cPLA2)), and bromoenol lactone (an inhibitor of the calcium-independent PLA2 (iPLA2)) inhibited the release of [3H]AA induced by ATP and Bz-ATP. In the absence of extracellular calcium, the release of [3H]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF3 and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF3. It is concluded that the P2X7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA2 and a calcium-independent iPLA2.

Localization of AMPA-selective glutamate receptor subunits in the adult cat visual cortex.

We have studied the presence and distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-selective glutamate receptor subunits (GluR1, 2, 3, and 4) in the adult cat visual cortical areas 17, 18, 19, and the lateral suprasylvian areas (LSA). Reverse transcription-polymerase chain reaction (RT-PCR) amplification indicated that the genes encoding GluR1, 2, 3, and 4 are expressed in these areas and Western blot analysis revealed that the size of the corresponding peptides is similar to those described in the rat brain. In situ hybridization (ISH) using digoxigenin-labeled riboprobes showed that mRNAs coding for GluR1 and GluR3 were located in cells in all layers of the areas examined and also in the underlying white matter. GluR1 mRNA was relatively abundant throughout layers II-VI while GluR3 mRNA revealed a more laminated pattern of expression, preferentially labeling cells in layers II, III, V, and VI. The distribution of AMPA-selective receptor subunit peptides was studied by immunohistochemistry using subunit specific antibodies and found to be consistent with ISH results. In addition, we observed that most of the cells strongly labeled by the anti-GluR1 antibody were non-pyramidal neurons and that intense GluR2/3 immunoreactivity was seen preferentially in pyramidal neurons. Interestingly, double-labeling experiments indicated that neurons expressing gamma-aminobutyric acid (GABA) as well as the GluR1 subunit were particularly abundant in deeper layers. The GluR4 peptide was predominantly found in a relatively low number of layer III and layer V neurons with either pyramidal or non-pyramidal morphology. Finally, the distribution of neurons expressing the various receptor subunits was similar in all the visual cortical areas studied. These findings indicate a high expression of GluR1-3 subunits in the cat visual cortex and that GluR1 and GluR2/3 subunits are particularly abundant in non-pyramidal and pyramidal neurons, respectively. In addition, the results described here provide a reference for future studies dealing with the effect of visual deprivation on the expression of this receptor type.

Postoperative analgesia following total hip replacement: a comparison of intrathecal morphine and diamorphine.

Sixty patients undergoing elective total hip replacement under spinal anaesthesia were randomly assigned to receive either intrathecal (IT) diamorphine 0.75 mg (n = 30) or IT morphine 1.0 mg (n = 30). Postoperative pain scores, analgesic requirements and side effects were assessed by a blinded observer. Postoperative pain scores were broadly similar and satisfactory for both groups but the amount of additional IV morphine required to achieve this was significantly reduced in the morphine compared with the diamorphine group (P < 0.05). Twelve of the morphine group required no postoperative analgesics compared with four in the diamorphine group (P < 0.02). There were no differences between the groups in the incidence of side effects such as emesis and pruritus. No significant postoperative respiratory depression was noted. In the doses used intrathecal morphine provided superior postoperative analgesia to that of intrathecal diamorphine.

Intramuscular ketorolac following total hip replacement with spinal anaesthesia and intrathecal morphine.

We have studied the analgesic and morphine sparing effect of ketorolac tromethamine in 60 patients after total hip replacement under spinal anesthesia. In this double blind study 30 patients received ketorolac 30 mg IM 6 hourly postoperatively and the control group received saline. Analgesia was assessed by visual analogue pain scores (VAS) and morphine consumption by patient controlled analgesia (PCA). There was a significantly (P < 0.02) lower morphine consumption in the ketorolac group (7.1 +/- 8.6 mg; Mean +/- s.d.) when compared to the saline group (14.2 +/- 13.6 mg). Although there was a trend for lower VAS on the first postoperative night this was only significant at 10 hours postoperatively and the next morning at 08:00 hr. The incidence of side effects (emetic sequelae, pruritus and headache) were similar in both groups. It is concluded that ketorolac reduces the consumption of additional morphine in conjunction with intrathecal morphine but had no effects on the side effects.

Comparison of the analgesic effects of intrathecal clonidine and intrathecal morphine after spinal anaesthesia in patients undergoing total hip replacement.

We have studied the anaesthetic and analgesic properties of intrathecal clonidine and intrathecal morphine in patients undergoing total hip replacement under spinal anaesthesia. After routine spinal anaesthesia with 0.5% plain bupivacaine 2.75 ml, patients were allocated randomly to receive intrathecal clonidine, morphine or saline (control) as adjuvant to the bupivacaine. Postoperative analgesic effects were measured by consumption of morphine via patient-controlled analgesia and visual analogue pain scores. Both intrathecal clonidine and intrathecal morphine prolonged the time to first analgesia compared with saline (mean 278 (SD 93.2) min, 498 (282.4) min and 54 (61.9) min, respectively) (P < 0.001). Total morphine consumption on the first night after operation was significantly less in the intrathecal morphine group. There were no differences between the clonidine and the control group. Intrathecal clonidine prolonged the duration of spinal analgesia, but was markedly inferior to the intrathecal morphine in providing subsequent postoperative analgesia.

Intraoperative bupivacaine diminishes pain after lumbar discectomy. A randomised double-blind study.

A randomised double-blind study was carried out on 60 patients undergoing elective lumbar discectomy. Patients in the study group (n = 30) received an injection of 10 ml of 0.5% bupivacaine into the wound; the control group (n = 30) received none. Postoperative pain was measured by a visual analogue pain scale and by the amount of morphine administered by a patient-controlled analgesia system. Patients in the study group had lower pain scores, used less morphine, waited longer until their first demand for analgesia and reported their postoperative pain to be less severe.

Changes in blood flow and vascular resistance in the arm and leg during spinal anaesthesia and vasopressor therapy with methoxamine.

The effects of subarachnoid block with hyperbaric bupivacaine 0.5% on forearm and calf blood flow and vascular resistance were examined using venous occlusion plethysmography in 10 fit patients. Following blockade there was an increase in mean (SD) calf blood flow from 2.0 (0.4) to 2.6 (1.2) ml.100 ml-1.min-1 (p = 0.057), a decrease in mean (SD) calf vascular resistance from 55 (15) to 38 (14) R units (p < 0.01), a decrease in mean (SD) forearm blood flow from 3.1 (1.2) to 1.8 (0.9) ml.100 ml-1.min-1 (p < 0.01) and an increase in mean forearm vascular resistance from 38 (16) to 62 (24) R units (p < 0.01). Methoxamine 2 mg was administered intravenously when the mean arterial blood pressure decreased by 15%. There was a resultant marked increase in mean (SD) forearm blood flow from 1.8 (0.9) to 3.0 (0.8) ml.100 ml-1.min-1 (p < 0.001) but a decrease in mean (SD) calf blood flow from 2.6 (1.2) to 2.1 (1.4) ml.100 ml-1.min-1 (p = 0.11). Correspondingly, the mean (SD) forearm vascular resistance decreased from 62 (24) to 46 (17) R units (p < 0.01) with an increase in mean (SD) calf vascular resistance from 38 (14) to 56 (22) R units (p < 0.01). These findings indicate that whereas methoxamine produces the anticipated constrictor response in the calf vessels it causes a simultaneous reduction in vascular resistance in the forearm.

The characteristics of analgesic requirements following subarachnoid diamorphine in patients undergoing total hip replacement.

BACKGROUND AND OBJECTIVES: The postoperative pain scores and analgesic requirements were assessed in 60 patients who had undergone total hip replacement under bupivacaine spinal anesthesia. METHODS: Thirty of the patients had intrathecal diamorphine injected after the bupivacaine, and the remaining 30 received saline. RESULTS: Superior postoperative pain relief was obtained in the diamorphine group, whose average postoperative morphine requirements were 12 +/- 11.4 mg compared to 31 +/- 18.7 mg (mean +/- SD) in the control group. Despite the lower doses of morphine, their pain scores over the first 24 hours postoperatively were consistently lower. No differences were seen between the groups with respect to respiratory depression, nausea, pruritus, postoperative sedation, headache, or urinary retention. CONCLUSION: Pain control after intrathecal diamorphine supplemented by intravenous morphine from a patient controlled analgesia system is superior to intravenous morphine alone.

Effects of omeprazole, with and without metoclopramide, in elective obstetric anaesthesia.

We report the results of a study comparing two dose regimens of the gastric antisecretory agent, omeprazole, used as prophylaxis against pulmonary aspiration of gastric contents during general anaesthesia for elective Caesarean section. Since antisecretory agents do not clear stomach contents already present at the start of treatment, two groups of patients who had received both omeprazole and a prokinetic drug, metoclopramide, were also studied. Thirty patients received oral omeprazole 40 mg on the evening before and on the morning of the operation (group 1), 33 received oral omeprazole 80 mg on the morning of the operation (group 2), and 15 (group 3) and 16 (group 4) patients respectively received the oral omeprazole doses stated above and in addition metoclopramide 10 mg given intramuscularly at least 20 min before induction of anaesthesia. Gastric aspirate pH and volume were measured at induction of anaesthesia and on completion of surgery. At induction of anaesthesia, treatment was judged successful (pH > or = 2.5 and volume < 25 ml) in 87%, 73%, 100% and 81% of patients in groups 1-4 respectively. The corresponding results on completion of surgery were 100%, 88%, 100% and 100%. While omeprazole is useful as prophylaxis against pulmonary aspiration during general anaesthesia for elective Caesarean section, the addition of a prokinetic agent seems to be necessary to maximise its effects.

Propofol and serum potassium.

The aim of this study was to examine the effect of propofol and propofol with suxamethonium on serum potassium concentration during induction of general anaesthesia. Forty patients were studied during elective surgery and serum electrolytes were measured before and after the induction of anaesthesia. Patients were allocated at random into two groups to receive either propofol alone or with suxamethonium. The serum potassium fell by 0.04 mmol.l-1 by 5 min after induction with propofol alone but had recovered to the pre-induction level by 10 min. The serum potassium rose by 0.23 mmol.l-1 when a combination of propofol and suxamethonium was used for the induction of anaesthesia.