The platelet aggregometer and beyond: Gustav Born at the Royal College of Surgeons.

In 1960, Gustav Born was appointed to head the Department of Pharmacology at the Royal College of Surgeons in London. The next 13 years  would prove to be  the most productive in his scientific career and the most important in the development of the Department into an internationally respected research center. The advances in platelet biology were made possible by the evolution of the platelet aggregometer, brilliantly conceived and developed by Born and his team, into a robust and reliable scientific instrument, with which they, quite literally, revolutionized the study of platelet function. For the first time, the actions of agonists and antagonists could be quantified pharmacologically, and the biochemistry of aggregation analyzed, identifying two systems, cyclic nucleotides and phospholipid metabolism, as crucial to the understanding of the pathophysiology of human platelets. Recognizing the critical importance of the interplay between platelets, leucocytes and the vascular endothelium, in the formation and in the rupture of atheromatous plaques, his group also investigated aggregation in the microvasculature in vivo. Born's never-ending flow of ideas and enthusiasm for their exploration created an atmosphere of discovery in his group that matched that of his colleague, John Vane. It was a collaboration between these two teams that elucidated the mechanism of action of the non-steroidal anti-inflammatory drugs (NSAIDs) and established the prophylactic use of aspirin as an anti-thrombotic therapy - indeed, two of the most significant pharmacological discoveries of the twentieth century.

Sérgio Ferreira and Bothrops jararaca at the Royal College of Surgeons, London.

In 1965, Sérgio Ferreira had completed his PhD programme under the supervision of Prof Rocha e Silva, his thesis had been accepted, and he was preparing to go to England for his first post-doctoral fellowship at the Pharmacology Department at Oxford University [...].

Endogenous opioid and cannabinoid mechanisms are involved in the analgesic effects of celecoxib in the central nervous system.

BACKGROUND/AIMS: In this study we analyzed the mechanisms underlying celecoxib-induced analgesia in a model of inflammatory pain in rats, using the intracerebroventricular (i.c.v.) administration of selective opioid and cannabinoid antagonists. METHODS AND RESULTS: Analgesic effects of celecoxib were prevented by selective µ-(ß-funaltrexamine) and δ-(naltrindole), but not κ-(nor-binaltorphimine) opioid antagonists, given i.c.v. 30 min before celecoxib. Similar pretreatment with AM 251, but not SR 144528, cannabinoid CB(1) and CB(2) receptor antagonists, respectively, prevented celecoxib-induced analgesia. The fatty acid amide hydrolase inhibitor, URB 597, also prevented celecoxib-induced analgesia. CONCLUSIONS: Our data provided further evidence for the involvement of endogenous opioids and revealed a new cannabinoid component of the mechanism(s) underlying celecoxib-induced analgesia.

Is the sulphonamide radical in the celecoxib molecule essential for its analgesic activity?

In a model of peripherally induced inflammatory pain in rats, selective inhibitors of cyclooxygenase (COX)-2 raised nociceptive thresholds above basal values, an effect referred to as "hypoalgesia". However other, non-selective, inhibitors of COX (indomethacin, piroxicam) or a selective inhibitor of COX-1 did not induce hypoalgesia in this model, implying that COX inhibition was not causally related to the hypoalgesic effect. Here, we have assessed whether other COX-2 inhibitors or other sulphonamides, apart from celecoxib could exhibit hypoalgesia in our model of inflammatory pain. Inflammation was induced in one hind paw of rats by intraplantar injection of carrageenan (250 µg). Nociceptive thresholds to mechanical stimulation were measured in the inflamed and contralateral paws for 6 h after carrageenan. Three sulphonamides, celecoxib itself, furosemide (a loop diuretic), acetazolamide (a carbonic anhydrase inhibitor), or a selective COX-2 inhibitor lacking the sulphonamide group, lumiracoxib, were injected s.c., 30 min before the pro-inflammatory stimulus. Naltrexone, a non-selective opioid antagonist was also administered s.c., 30 min before test drugs. Furosemide and acetazolamide dose-dependently induced hypoalgesia in the inflamed paw, as did celecoxib. However, lumiracoxib only produced anti-hyperalgesia. Pre-treatment with naltrexone completely prevented the hypoalgesia induced by the sulphonamides, but only partially prevented the anti-hyperalgesic effect of lumiracoxib. Taken together, our results suggest that the sulphonamide group in the structure of celecoxib is more critical for the development of hypoalgesia in our model than its ability to inhibit COX-2. Further, other sulphonamides lacking significant COX inhibition were also able to exhibit hypoalgesic effects, mediated by the endogenous opioid system.

How ACE inhibitors transformed the renin-angiotensin system.

The renin-angiotensin system (RAS) now underlies the successful treatment of almost 50% of the patients in cardiovascular medicine, with serious possibilities of extension to diabetes, Alzheimer's disease and cancer. This clinical transformation started just over 50 years ago, with the unexpected identification of a bradykinin-potentiating peptide from snake venom, as a potent inhibitor of ACE which led to the development of the first synthetic inhibitor, captopril, followed by the angiotensin receptor blockers. This article analyses the transformation of the RAS into its different stages, from academic experiments to clinical use and back to the laboratory, identifying the critical events involved, both clinical and scientific. The analysis also assesses the contributions of chance, coincidence, and conviction that were crucial in this transformation. Although questions remain, the transformation of the RAS over the past five decades provides a success story for medicine, for pharmacology, and, most significantly, for patients.

The Second Gaddum Lecture: its origins and outcomes.

Fifty years ago, the BJP published the Second Gaddum Lecture, given by John Vane to the British Pharmacological Society. This article assesses the origins of the experiments described in the Lecture, linking them directly to Gaddum's use of bioassay, a defining feature of pharmacology. The outcomes of those experiments are also assessed, tracking those results that have survived the past five decades. Two of the major advances in cardiovascular medicine, the ACE inhibitors, as anti-hypertensives, and low-dose aspirin, to prevent thrombosis were initiated by the work in this Lecture. Physiologically significant outcomes include a new non-respiratory function of the lung, based on the metabolism of endogenous vasoactive substrates in the pulmonary circulation and the recognition of the endothelium as a highly interactive component of blood vessels. The present state of the art in pharmacology, physiology and medicine owes much to the work described in the Second Gaddum Lecture.

Different mechanisms underlie the analgesic actions of paracetamol and dipyrone in a rat model of inflammatory pain.

BACKGROUND AND PURPOSE: The analgesics, paracetamol and dipyrone are weak inhibitors of the cyclooxygenase isoforms 1 or 2 (COX-1, COX-2) but more potent on COX-3. Both are also weak anti-inflammatory agents, relative to their analgesic and antipyretic activities. In a model of inflammatory pain mediated by prostaglandins, both compounds were analgesic. We have analysed this shared effect further in order to elucidate the underlying mechanisms. EXPERIMENTAL APPROACH: Inflammation was induced in one hind paw of rats by intraplantar injection of 250 microg lambda-carrageenan (CG) and the contralateral paw injected with saline. Nociceptive thresholds to mechanical stimulation were measured immediately before and for 6 h after, injection of CG. The analgesics were s.c. or locally (intraplantar) injected either 30 min before or 2 h after CG. In some groups, naltrexone was injected (s.c. or intraplantar), 1 h before CG. KEY RESULTS: Pretreatment with paracetamol or dipyrone (60-360 mg kg(-1)) reversed hyperalgesia induced by CG and increased nociceptive threshold in the inflamed paw above the basal level (hypoalgesia). Paracetamol, but not dipyrone, also raised nociceptive thresholds in the non-inflamed paw. Subcutaneous, but not local, administration of naltrexone, a specific opioid antagonist, reversed the hypoalgesia induced by paracetamol, but similar naltrexone treatment had no effect on dipyrone-induced analgesia. CONCLUSIONS AND IMPLICATIONS: Although both paracetamol and dipyrone are inhibitors of COX isoforms and thus of prostaglandin biosynthesis and were analgesic in our model, their analgesic actions were functionally and mechanistically different. Satisfactory mechanisms of action for these analgesics still remain to be established.

Calcitonin-gene related peptide is a potent inducer of oedema in rat orofacial tissue.

BACKGROUND AND AIMS: This study aimed to assess the potential of calcitonin-gene related peptide (CGRP), a neuropeptide released from sensory nerves, to induce oedema in orofacial tissue. EXPERIMENTAL APPROACH: Wistar rats (150-200 g) anesthetized with isoflurane were injected intraorally with CGRP (100 µl; 8-33 pmol) in the right side of the mouth. The contralateral side was injected with the same volume of physiological saline. Increased cheek thickness (in mm), as a measure of oedema formation, was assayed bilaterally with a digital caliper before (T = 0) and up to 24 h following injection of CGRP. Pretreatment with antagonists (CGRP8-37, 10 nmol; pizotifen, 2 mg/kg) was given by intra-oral or subcutaneous injection, 10 or 30 min, respectively, before the inflammatory stimulus. CGRP and CGRP8-37 were also injected into the rat hind paw to induce oedema. Data are presented as the mean (±SEM) difference in thickness between the right and the left sides at each time. RESULTS: Following intra-oral injection, CGRP induced a rapidly developing (5-15 min) and long-lasting (6 h), dose-dependent oedema in the rat cheek, blocked by pre-treatment with CGRP8-37 or pizotifen. CGRP induced a smaller oedematogenic effect in the rat hind paw also blocked by the CGRP antagonist. CGRP (16 pmol) potentiated the oedema induced by co-injected substance P (3.7 nmol) and contributed to the oedema following intraoral injection of carrageenan (100 µg). Injection of CGRP8-37 alone induced an early but short-lasting oedema. CONCLUSION: Local injection of CGRP potently induced oedema in the orofacial tissue of rats which was blocked by a CGRP receptor antagonist. The overall inhibition of carrageenan-induced oedema by CGRP8-37 suggests that endogenous CGRP contributes to an oedematogenic response in orofacial tissues.

Tumor growth, angiogenesis and inflammation in mice lacking receptors for platelet activating factor (PAF).

Tumor growth is associated with angiogenesis and inflammation and the endogenous lipid, platelet activating factor (PAF), is a pro-inflammatory and pro-angiogenic mediator. We therefore measured tumor growth, angiogenesis and inflammation in normal (WT) mice and those lacking the receptor for PAF, through gene deletion (PAFR-KO). Growth of solid tumors derived from colon 26 cells was not altered but that from Ehrlich cells was markedly (5-fold) increased in the PAFR-KO mice, relative to the WT strain. Angiogenesis, as tumor content of VEGF or hemoglobin, was increased in both tumors from the mutant strain. Inflammation, as neutrophil and macrophage accumulation and chemokine (CXCL2 and CCL2) content of tumors, was decreased or unchanged in the tumors implying an overall decrease in the inflammatory response in the PAFR-KO strain. We also assessed growth of the Ehrlich tumor in its ascites form, after i.p. injection. Here growth (ascites volume) was inhibited by about 30%, but neutrophil and macrophage numbers were increased in the ascites fluid from the PAFR-KO mice. Angiogenesis in the peritoneal wall, which is not invaded by the tumor cells, was increased but leukocyte infiltration decreased in the mutant strain. Our results show, unexpectedly, that tumor-induced angiogenesis was increased in mice lacking response to PAF, from which we infer that in normal (WT) mice, PAF is anti-angiogenic. Further, although growth was still associated with angiogenesis in PAFR-KO mice, growth was not correlated with inflammation (leukocyte accumulation).

Host response to sponge implants differs between subcutaneous and intraperitoneal sites in mice.

Synthetic matrices have been used widely to repair and/or to replace biological tissues. However, there is relatively little information on the effect of different anatomical compartments on the host response to foreign implants. We have analyzed such responses to sponge implants in subcutaneous and in intraperitoneal sites in mice at days 3, 5, and 8 postimplantation by measuring inflammation, angiogenesis, and production of proangiogenic/inflammatory cytokines. The angiogenic response, assessed by hemoglobin content and by morphometric analysis of the number of vessels, was higher in intraperitoneal implants. Levels of vascular endothelial growth factor in intraperitoneal implants were 14-fold higher than in subcutaneous implants at day 3 and remained high for the next 5 days. Neutrophil accumulation as determined by myeloperoxidase activity was the same in both types of implants. Macrophage accumulation (N-acetylglucosaminidase activity) was also similar on days 3 and 8 in both implants. Levels of the chemokine CXCL2/KC were always higher, but those of CCL2/JE lower, in the intraperitoneal implant. These results demonstrate that the anatomical site of the implant markedly influenced the host response to synthetic matrices. Our results provide a greater understanding of factors affecting the biocompatibility of exogenous materials placed at different anatomical sites.

Ketamine-xylazine anaesthesia and orofacial administration of substance P: A lethal combination in rats.

BACKGROUND AND AIMS: Ketamine+xylazine mixture is a widely used anaesthetic in animal experiments. In rats anaesthetized with this mixture, we have shown that injection of carrageenan, a standard proinflammatory stimulus, into the cheek (intra-oral injection) induced oedema. A likely mediator of this oedema is substance P (SP), a major transmitter of sensory nerves in orofacial tissue. We have assessed the effects of intra-oral injection of SP in rats. EXPERIMENTAL APPROACH: SP (50-1µg per rat) was injected intra-orally in male adult Holtzman or Wistar rats, anaesthetized with ketamine+xylazine. For comparison, histamine (50µg) and 5-HT (5µg) were similarly injected. Antagonists of SP (SR140333, 2mg/kg), of histamine (pyrilamine, 2mg/kg) or of 5-HT (pizotifen, 2mg/kg) were subcutaneously (s.c.) injected, 30min before the corresponding agonist. Oedema in the cheek was assessed by measuring tissue thickness with calipers. RESULTS: Intra-oral injection of SP (1-50µg per rat) in Holtzman or Wistar rats anaesthetized with ketamine+xylazine induced, dose-dependently, death within 15min, accompanied by signs of excessive salivation. Rats pretreated with SR140333 were protected against SP-induced lethality and the excessive salivation. However, intra-oral injection of either histamine or 5-HT did not induce death, only a characteristic cheek oedema. These doses of SP injected into the hindpaws of conscious Holtzman and Wistar rats only induced oedema with no deaths. In rats anaesthetized with inhaled isoflurane, intra-oral SP (50µg) induced only cheek oedema, with no deaths or excessive salivation. This oedema was prevented by pre-treating rats with SR140333, pyrilamine and pizotifen. CONCLUSION: It is likely that the deaths were due to excessive salivation induced by the particular combination of ketamine and SP. Our results are presented as a warning to other experimenters who might use these two otherwise non-toxic conditions and the consequent unexpected and needless loss of experimental animals.

Endogenous opioids mediate the hypoalgesia induced by selective inhibitors of cyclo-oxygenase 2 in rat paws treated with carrageenan.

Mechanical hyperalgesia induced in rat paws by carrageenan (250microg) was modified by pre-treatment with three selective inhibitors of cyclo-oxygenase-2 (COX-2); celecoxib, rofecoxib and SC236. These inhibitors raised the nociceptive threshold above the normal, non-inflamed, level, inducing a state of hypoalgesia. Such hypoalgesia was observed in different strains of rat (Holtzman, Wistar and Sprague-Dawley) and after different modes of administration of the COX-2 inhibitor (locally, in the paw, or systemically). A selective inhibitor of COX-1 (SC 560; 1-10mg kg(-1)) decreased hyperalgesia but did not induce hypoalgesia. Pre-treatment with naltrexone (3mg kg(-1)), an opioid receptor antagonist, did not affect carrageenan-induced hyperalgesia but abolished the hypoalgesic effects of COX-2 inhibitors, without diminishing the anti-hyperalgesic effect of indomethacin. In rats made tolerant to the anti-nociceptive effects of morphine, all anti-nociceptive effects of SC236 were abolished but the anti-hyperalgesic effects of indomethacin or SC 560 were unaffected. We conclude that, in our model of inflammatory hyperalgesia, the anti-nociceptive effect of selective COX-2 inhibitors involved the participation of endogenous opioids.

Monoamine oxidase: isoforms and inhibitors in Parkinson's disease and depressive illness.

A few years after the foundation of the British Pharmacological Society, monoamine oxidase (MAO) was recognized as an enzyme of crucial interest to pharmacologists because it catalyzed the major inactivation pathway for the catecholamine neurotransmitters, noradrenaline, adrenaline and dopamine (and, later, 5-hydroxytryptamine, as well). Within the next decade, the therapeutic value of inhibitors of MAO in the treatment of depressive illness was established. Although this first clinical use exposed serious side effects, pharmacological interest in, and investigation of, MAO continued, resulting in the characterization of two isoforms, MAO-A and -B, and isoform-selective inhibitors. Selective inhibitors of MAO-B have found a therapeutic role in the treatment of Parkinson's disease and further developments have provided reversible inhibitors of MAO-A, which offer antidepressant activity without the serious side effects of the earlier inhibitors. Clinical observation and subsequent pharmacological analysis have also generated the concept of neuroprotection, reflecting the possibility of slowing, halting and maybe reversing, neurodegeneration in Parkinson's or Alzheimer's diseases. Increased levels of oxidative stress in the brain may be critical for the initiation and progress of neurodegeneration and selective inhibition of brain MAO could contribute importantly to lowering such stress. There are complex interactions between free iron levels in brain and MAO, which may have practical outcomes for depressive disorders. These aspects of MAO and its inhibition and some indication of how this important area of pharmacology and therapeutics might develop in the future are summarized in this review.

Phospholipases A2 and C of human lung; subcellular distribution and substrate selectivity.

The phospholipase activities of cell-free extracts of human lung were studied using sn-2-arachidonoyl phospholipids. Samples of human lung obtained during surgery were homogenized and separated by centrifugation into three fractions: P1, containing mitochondrial and lysosomal marker enzymes; P2, with microsomal enzymes; and S2, with cytosolic enzymes. The highest phospholipase activities were in the microsomal fraction, using any of the three substrates, [14C]arachidonoylphosphatidylcholine (PC), [14C]arachidonoylphosphatidylethanolamine (PE) and [14C]arachidonoylphosphatidylinositol (PI). From PC and PE, only free arachidonic acid was formed, suggesting the presence of a phospholipase A2 (PLA2)-like activity. From PI, two metabolites were produced, diacylglycerol and arachidonic acid, suggesting the presence of a PI-specific PLC activity. Rates of hydrolysis were highest for PI, followed by PE and then PC. Hydrolysis of [14C]arachidonoyl-PC was compared to that of [14C]oleoyl-PC and found to be similarly distributed and of comparable velocity. The distribution and relative activities of phospholipases in rat lung homogenates were very similar to those in human lung.

Decreased developmental cell death in sympathetic and spinal sensory nervous systems of the Kyoto spontaneously hypertensive rat.

OBJECTIVE: To investigate the hypotheses that Kyoto spontaneously hypertensive rats (SHR) possess more sympathetic neurons than do normotensive Wistar-Kyoto (WKY) animals due to reduced perinatal cell death and that this is due to increased availability of the sympathetic survival neurotrophin, nerve growth factor. METHODS: Total cell counts of neuron numbers were performed in neonatal and adult SHR and WKY rat superior cervical ganglia and correlated with counts of apoptotic cells. The values for sympathetic neuron numbers were compared with those for a spinal sensory ganglion. Immunocytochemistry was used to obtain more information about the phenotypes of neurons counted. RESULTS: Adult SHR sympathetic ganglia contained about 25% more sympathetic neurons than did those of WKY animals. Similar elevation of numbers was found both for neurons containing and for those devoid of neuropeptide Y. In neonatal animals, in contrast, there was no strain difference in sympathetic cell numbers but the number of apoptotic cells was reduced in SHR. Spinal sensory neuron numbers in adult SHR were elevated to a similar extent as were sympathetic neurons, but biochemical and morphometric data suggested that this change does not involve cells that are sensitive to nerve growth factor. CONCLUSIONS: Although our results support the view that there is reduced developmental cell death both in sympathetic and in sensory systems, they also suggest that this is unlikely to be due to a simple excess of nerve growth factor during development.

Pulmonary metabolism of bradykinin analogues and the contribution of angiotensin converting enzyme to bradykinin inactivation in isolated lungs.

1 The activity and pulmonary metabolism of two peptides, 7-homo Pro-bradykinin and 8-homo Phe-bradykinin were studied in isolated systems. 2 Both analogues were about 50-70 times less active than bradykinin on the guinea-pig ileum and 70-160 times less active on isolated strips of cat terminal ileum. 3 The action of both analogues on guinea-pig ileum was potentiated (2.5-3.0 fold) by a bradykinin potentiating peptide (BPP9a) but less so than the action of bradykinin (4-5 fold). 4 Like bradykinin, the 8-homo Phe analogue was extensively inactivated (greater than 90%) in a single passage through the pulmonary circulation of guinea-pig or rat isolated lungs and this inactivation was prevented by pre-treatment of the lungs with BPP9a. 5 The 7-homo Pro analogue was inactivated to a lesser degree in guinea-pig lungs (58%) and in rat lungs (89%) and its inactivation was not affected by BPP9a. 6 It is concluded that the 8-homo Phe analogue is a substrate for the dipeptidylcarboxypeptidase (angiotensin I converting enzyme) of lung, whereas the 7-homo Pro analogue is not a substrate. 7 There is about four times as much dipeptidylcarboxypeptidase activity in guinea-pig isolated lungs as there is in rat isolated lungs.

Inhibition of angiotensin I converting enzyme by venom peptides.

The synthetic analogues of three naturally occurring peptides from the venom of Bothrops jararaca have been tested as inhibitors of angiotensin converting enzyme activity in two systems. They have widely differing absolute potencies but the relative potencies are similar in the two systems. The high inhibitory potency of the nonapeptide is compatible with a non-competitive type of inhibition.

Inhibition by clinically used dyes of prostaglandin inactivation in rat and human lung.

1. The effect of several clinically used dyes on prostaglandin E2 (PGE2) inactivation was studied in rat and human isolated lung. 2. All the dyes, given as infusions through the pulmonary circulation, inhibited the inactivation of PGE2 as measured by bioassay. The action of the dyes was readily reversible on stopping the infusion. 3. The concentration producing 30% inhibition varied from 0.6 microM for indocyanine green to over 100 micro M for methylene blue. 4. Inhibition of PGE2 inactivation was also observed when the dyes were given as bolus injections during PGE2 infusions through the lung. 5. These dyes are therefore not physiologically inert as they had previously been thought to be and their interaction with prostaglandins may affect the variables they are used to measure.

Action of prostaglandin dehydrogenase inhibitors on prostaglandin uptake in rat isolated lung.

1. The effects of some inhibitors of 15-hydroxy-prostaglandin dehydrogenase (PGDH) have been studied on the inactivation of prostaglandin E2 (PGE2) and 16, 16-dimethyl PGE2 in rat isolated lung. 2. Bioassay was used to show that the inactivation of PGE2 and its methyl analogue, was inhibited by frusemide (10(-3) M), N-ethyl maleimide (10(-5) M) and 5,5'-dithio-bis-2-nitro-benzoic acid (10(-2) M), but not by caffeine (2.5 x 10(-3) M). 3. The efflux of radioactivity from lung following injection of [14C]-PGE2 was faster in lungs treated with frusemide, N-ethyl maleimide or bromcresol green, than in untreated lungs. 4. Caffeine (10(-3), 10(-2) M) did not change the rate of 14C-efflux from lungs following injection of [14C]-PGE2. 5. From these results it is concluded that those PGDH inhibitors that prevented prostaglandin inactivation in isolated lungs did so by inhibiting uptake of prostaglandin rather than by inhibiting PGDH.