Immunohistochemical localization of substance P in postmortem rat and human spinal cord.

The stability of substance P-like immunoreactivity was examined in postmortem rat and human spinal cord using radioimmunoassay and indirect fluorescence immunohistochemistry. The distribution of fluorescence in rat and human spinal cord was unchanged at intervals up to 48 hours (h) and 87 h, respectively. Fine linear fluorescent fibers were seen only in rat spinal cord processed at time intervals up to two h postmortem; they were never observed in human spinal cord, which was routinely obtained more than 6 h postmortem. The content of substance P-like material in human spinal cord nd its immunohistochemical appearance was not affected by age, the nature of the terminal illness, or autopsy delay. However, no correlation was found between intensity of fluorescent staining and content as measured by radioimmunoassay. It may be possible to use postmortem analysis of substance P in diseases of the central nervous system, but radioimmunoassay and immunohistochemistry should be used concurrently.

The effect of age on A delta- and C-fibre thermal pain perception.

It has been suggested that ageing may have a differential effect on C fibre-mediated protopathic/tonic pain versus epicritic/phasic pain perception mediated by A delta fibres. The present study attempted to independently assess age-related changes in the function of A delta- and C-nociceptive fibres by examining CO2 laser-induced thermal pain thresholds before, during and after a compression block of the superficial radial nerve in 15 young and 15 healthy elderly adult subjects. Nerve block efficacy was monitored via measures of cold, warm and mechanical threshold, and simple reaction time. During nerve compression block, reaction time and mechanical threshold increased, cold sensation became impaired while warm sensation remained unaffected throughout the test in both groups. With respect to pain sensitivity, young adults exhibited significant increases in thermal pain threshold during A-fibre block while pain threshold remained relatively stable across the 3 test periods in the elderly group. It would appear that elderly adults rely predominantly on C-fibre input when reporting pain whereas younger adults utilise additional input from A delta fibres. Subsequent analysis revealed that during pre- and post-block periods, older adults exhibited a significant elevation in thermal pain threshold; however, when A delta-fibre function was impaired and only C-fibre information was available, both groups responded similarly. These findings support the notion of a differential age-related change in A-fibre-mediated epicritic pain perception versus C-fibre-mediated protopathic pain.

Altered heat pain thresholds and cerebral event-related potentials following painful CO2 laser stimulation in subjects with fibromyalgia syndrome.

A decrease in mechanical pressure pain thresholds, particularly over pre-designated tender points, is one of the defining characteristics of fibromyalgia syndrome (FS); however, changes in thermal pain sensitivity have not been investigated. The present study examined heat pain thresholds and cerebral event-related potentials following CO2 laser stimulation in 10 subjects with FS and 10 age-matched control volunteers. The results indicate that patients with FS exhibit a significant reduction in heat pain threshold when tested on the dorsal surface of the hand. In accordance with previous research, we also found a decrease in mechanical pain threshold over pre-designated tender points and at control sites as well as a significantly larger mechanically induced neurogenic flare response. These measures were highly correlated with thermal pain threshold even though different anatomical sites were stimulated. Hence, it seems likely that FS patients display a multimodal change in pain sensitivity which is generalized rather than anatomically restricted. Patients with FS also displayed a significant increase in the peak-to-peak amplitude of the cerebral potential evoked by CO2 laser stimulation at pain threshold intensity and 1.5 times pain threshold intensity. These findings suggest a greater activation of central nervous system (CNS) pathways following noxious input. Putative explanations for the increased CNS response are discussed, including mechanisms of peripheral nociceptor sensitization, altered CNS function and the role of psychological factors.

Age-related differences in the time course of capsaicin-induced hyperalgesia.

The effect of age on hyperalgesia, one of the most common signs of injury, has not been previously examined in humans. A psychophysical study was conducted in 10 young (26.9+/-4.6 years) and 10 older (79. 0+/-5.7 years) healthy volunteers to investigate the effect of age on the development of hyperalgesia induced by topical application of capsaicin (0.1 ml, 5 mg/ml). The capsaicin patch (diameter 2 cm) was applied for 1 h. The intensity of capsaicin-induced spontaneous sensation, mechanical pain threshold, area of flare, heat and punctate hyperalgesia were measured hourly for 3 h after the application. Older adults took a longer period to report first pain. There was no age effect on the magnitude of spontaneous sensation, flare size and area of heat hyperalgesia. The area of heat hyperalgesia rapidly decreased over time in both age groups. In marked contrast, the area of punctate hyperalgesia and associated reduction in the mechanical pain threshold were maintained in older adults over the entire 3 h test period, but resolved rapidly in young adults. We conclude that, given the same intensity of noxious stimulation, older adults display a similar magnitude of hyperalgesia as younger persons. However, once initiated, punctate hyperalgesia appears to resolve more slowly in older people. This finding may indicate age differences in the plasticity of spinal cord neurons following an acute injury.

Photoperiodic variation in substance P induced plasma extravasation in the rat.

Photoperiodic variation in plasma extravasation was examined following exogenous perfusion of substance P over a vacuum-induced blister base on the rat hind footpad. The results showed a generalised suppression of plasma extravasation during the dark or active phase of the light/dark cycle which appears to be independent of the circadian fluctuations in endogenous corticosterone. These findings emphasise the need to consider photoperiodic conditions as a possible source of variability in neurogenic inflammatory responses.

Tachykinin-mediated modulation of the primary antibody response in rats: evidence for mediation by an NK-2 receptor.

Evidence for the involvement of primary afferent nerves and their associated neuropeptides in in vivo immunologic responses has been based on experiments in rats in which destruction of primary afferent nerves by the sensory neurotoxin capsaicin results in a diminished ability of the animal to mount a primary antibody response to sheep red blood cell (SRBC) antigen. This effect was shown to be reversed by substance P infusion immediately following antigenic stimulation. In this report we show that neurokinin A (NKA) is 12 times more potent than substance P in its capacity to reverse the effects of neonatal capsaicin pretreatment on the antibody response. Neurokinin A has a pD2 of 6.65 compared to 5.98 for substance P. In addition, NKA was more potent than substance P in reversing the effects of surgical lesions 2 days prior to antigenic stimulation. The effects of the D- and L-Pro9 analogues of [Glp6, Pro9]-SP6-11 on the plaque-forming cell response in capsaicin-treated rats provide further support for the hypothesis that the tachykinin receptor modulating the primary antibody response is an NK-2 receptor. These results demonstrate, for the first time, a role for NKA in in vivo immunomodulation.

Effects of capsaicin treatment on immunoglobulin secretion in the rat: further evidence for involvement of tachykinin-containing afferent nerves.

Neonatal capsaicin treatment has previously been shown to diminish the primary antibody response of adult rats to the subcutaneously administered T-dependent antigen, sheep red blood cells, as measured using a modification of the Cunningham plaque-forming cell assay technique. We have now studied the kinetics of this response in adult normal, neonatally capsaicin-pretreated and neonatally capsaicin-pretreated substance P-infused rats, and examined the effects of the tachykinin antagonist Spantide, on the plaque-forming cell response. Capsaicin pretreatment did not affect the antigen-specific plaque-forming cell response over the first 4 days following antigen injection. At days 5, 6 and 7 of the response, there was a statistically significant decrease in the number of plaque-forming cells secreting antigen-specific IgM, an effect not observed in capsaicin-pretreated rats which were given a subcutaneous infusion of substance P at the time of antigen injection. The tachykinin antagonist Spantide inhibited the plaque-forming cell response in normal rats after in vivo infusion at the time of antigen injection by more than 70%. This effect of Spantide was dose dependent, occurred with maximal effect at 10 microM, and appeared to be independent of any histamine-mediated action. The results of this study provide further evidence for a receptor-mediated immunomodulatory role of tachykinin-containing primary afferent nerves.

Nitric oxide and sensory nerves are involved in the vasodilator response to acetylcholine but not calcitonin gene-related peptide in rat skin microvasculature.

1. The contributions of sensory nerves and nitric oxide (NO) to vasodilator responses to acetylcholine (ACh) and calcitonin gene-related peptide (CGRP) were examined in rat skin microvasculature with a laser Doppler flowmeter to monitor relative blood flow. 2. Perfusion of ACh (100 microM; for 30 min) over a blister base on the rat hind footpad elicited microvascular vasodilatation and this response was not sustained. CGRP (1 microM; 10 min perfusion) also elicited vasodilatation and this response was maintained even when CGRP was no longer in contact with the blister base. 3. The vasodilator response to ACh was significantly smaller in rats pretreated as neonates with capsaicin to destroy primary sensory afferents than it was in age-matched controls. The vasodilator response to CGRP was unaffected by capsaicin pretreatment. 4. Selective inhibitors of NO synthase, NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA) (both at 100 microM) attenuated the vasodilator response to ACh in control rats, but had no effect on the vasodilator response to CGRP. There was a significant L-NOARG-resistant component in control rats while in capsaicin-treated rats the vasodilator response to ACh was virtually abolished by L-NOARG. The inactive stereoisomer NG-monomethyl-D-arginine (100 microM) did not affect the vasodilator response to ACh. 5. The efficacy of L-NOARG and L-NMMA as inhibitors of endothelium-dependent responses was confirmed by use of an endothelium-dependent vasodilator, the calcium ionophore A23187 (100 microM; 10 min perfusion). Vasodilatation to A23187 was strongly attenuated by both L-NOARG and L-NMMA.6. These results suggest that sensory nerves and NO are both involved in the dilatation produced by ACh in rat skin microvasculature. A component of the vasodilator response elicited by ACh involves a direct action on the microvascular endothelium with subsequent generation of NO, while an additional component is elicited via activation of sensory nerves. The vasodilator mediator(s) released by ACh from sensory nerves acts largely independently of NO.7. The vasodilator response to CGRP is independent of a prejunctional action on sensory nerves and of NO.

NK-1 receptor mediation of neurogenic plasma extravasation in rat skin.

1. Plasma extravasation was induced by electrical nerve stimulation and by perfusion of tachykinins over a vacuum-induced blister base on rat footpad. 2. Stimulation of the sciatic nerve (18 V, 15 Hz, 0.5 ms) for 20 min produced a significant increase in the protein content of the perfusate. The response in capsaicin pretreated rats was only 4% of the control response. This indicates that the electrically-induced plasma extravasation response was mediated by capsaicin-sensitive sensory fibres. 3. Exogenous perfusion of the mammalian tachykinins substance P, neurokinin A and neurokinin B and the non-mammalian tachykinins physalaemin, kassinin and eledoisin was used to determine the tachykinin receptor type mediating the plasma extravasation response. Dose-response curves of the tachykinins (10(-9) M-10(-4) M) gave a rank order of potency of substance P = physalaemin greater than eledoisin greater than or equal to kassinin greater than neurokinin B = neurokinin A. 4. In addition, specific agonists of neurokinin receptors were perfused. Perfusion of [Glp6, D-Pro9] SP6-11 and [Glp6, L-Pro9]SP6-11 demonstrated that the L-Pro isomer was much more potent than the D-Pro isomer. 5. The rank order of potency and the greater potency of [Glp6, L-Pro9]SP6-11 over its D-isomer indicate an NK-1 neurokinin receptor mediates plasma extravasation in rat footpad skin.

Enkephalins and Endorphins. Clinical, pharmacological and therapeutic implications.

During the past 8 years there has been substantial progress in our understanding of the structure, distribution and action of endogenous opioid peptides. Currently, there appear to be 2 groups of peptides; those derived from beta-lipotropin and an enkephalin-related group. Some of these peptides have been shown to be distributed widely in the central nervous system and in endocrine tissues. The activity of the peptides probably occurs at several receptors but only 1 relatively specific (mu-receptor) antagonist, naloxone, is well studies. Although there have been many clinical studies of the action of opioids in man, no novel therapeutic applications have yet been established in clinical practice. Of the many areas of involvement attributed to opioids, those of analgesia, reproductive endocrinology, opiate dependence, and certain as yet undefined subtypes of major psychoses seem reasonably promising. Speculation surround opioid involvement in other disorders such as spinal trauma, septic shock, alcohol dependence, "functional' gastrointestinal disease, diabetes and asthma is of considerable interest but is supported by less clinical evidence. It seems that as research in opioids advances, the putative physiological opioid "spheres of influence' widen. At the same time, opioid mechanisms of action are being revealed to be more subtle and complex than previously thought. As a consequence, the expectations of rapid therapeutic application of opioid peptides and their antagonists are being modified and refined and realistic research strategies applied. In view of the work reviewed in this paper it seems reasonable to expect that such work will pay dividends in the not too distant future.

The effect of medical status on the activity level of older pain clinic patients.

OBJECTIVE: This project sought to assess the effect of disease status on the activity level of older people suffering from chronic pain. DESIGN: A retrospective comparison of groups defined by disease attributes. SETTING: Outpatient pain management clinic for older people. SAMPLE: The sample consisted of 115 patients, of a possible 144, aged between 52 and 91 years, who were assessed upon admission to a multidisciplinary pain management clinic. Subjects were allocated to groups for comparison on the basis of the diagnosis of their pain problem and the extent of coexistent medical problems. MEASUREMENTS: Groups were compared on scores of activity level using the Human Activity Profile, with and without pain (McGill Pain Questionnaire) and depressive symptom (Geriatric Depression Scale) scores as covariates. MAIN RESULTS: Both pain diagnosis and number of additional medical problems characterized groups that were distinguishable by level of activity. A musculoskeletal disorder was associated with greater impact on activity than either postherpetic neuralgia or pain associated with a psychiatric diagnosis. Less activity was also evident among the subjects with several additional medical problems. However, this effect did not operate independently of depressive symptoms. CONCLUSIONS: Disease status is a factor that rarely explains variations in the pain experience of young adult patients with chronic pain. The results from this study suggest that disease state does influence self-reported activity level in older people with chronic pain. The influence of medical status should be acknowledged as an important factor when assessing and managing the older patient with chronic pain.

Bone density, vitamin D nutrition, and parathyroid hormone levels in women with dementia.

OBJECTIVE: To determine whether patients with dementia have reduced bone mass, altered vitamin D, or parathyroid hormone status. DESIGN: Survey. SETTING: University hospital outpatient department. PARTICIPANTS: Twenty women with DSM-III-R mild dementia living in the community were compared with 40 cognitively normal community-dwelling women, matched for age, who had been recruited as part of studies in elderly twins. MEASUREMENTS: Bone density at the lumbar spine and neck of femur by dual-energy X-ray absorptiometry, intact serum PTH, and 25-hydroxyvitamin D levels. MAIN RESULTS: There was no significant difference in bone density between the subjects with mild dementia and the age- and sex-matched controls. The intact PTH (mean +/- SD) in the demented subjects was 4.9 +/- 2.1 pmol/L compared with 2.9 +/- 1.7 pmol/L in the twin controls (P < .01). The mean 25-hydroxyvitamin D in the demented subjects was 61 +/- 33 nmol/L, whereas it was 90 +/- 38 nmol/L in the twin controls (P < .01). CONCLUSIONS: We conclude that there were no significant differences in the bone density of community-dwelling women with mild dementia compared with normals. However, there were significant differences in parathyroid hormone and vitamin D levels between the two groups, suggesting that there is a high prevalence of subclinical hypovitaminosis D in demented women in the community.

Tachykinin-induced vasodilatation in rat skin measured with a laser-Doppler flowmeter: evidence for receptor-mediated effects.

Vasodilatation was induced by perfusion of the tachykinins substance P (SP), neurokinin A and neurokinin B and the analogues [Glp6, D-Pro9]SP-(6-11) and [Glp6, L-Pro9]SP-(6-11) over the base of vacuum-induced blisters on the rat footpad. Vasodilatation was measured as change in blood flow using a laser-Doppler flowmeter. The tachykinins induced vasodilatation in a dose-response manner with a threshold of approximately 3 pmol and pD2's of 6.48, 6.13 and 6.21 for SP, neurokinin A and neurokinin B respectively. The D- and L-Pro analogues of [Glp6, Pro9]SP-(6-11) also induced vasodilatation in a dose-dependent manner. The L-Pro analogue was more potent than the D-Pro analogue (D/L ratio of the EC50's = 21) which suggests the involvement of an NK-1 type receptor in the mediation of small vessel vasodilatation. The vasodilatation to SP was reduced by 64% and 59% in capsaicin- and antihistamine-pretreated animals respectively, demonstrating the involvement of capsaicin-sensitive primary afferent nerves and mast cells in the vasodilatation component of the neurogenic inflammatory response.

Modulation of a peripheral inflammatory response to substance P by locally administered opioid receptor agonists.

Using a blister model of inflammation in the rat hind footpad, the present study was undertaken to examine possible peripheral effects of specific mu (DAGO) and delta (DSLET) opioid receptor agonists on an inflammatory response induced by substance P, the putative mediator of neurogenic inflammation. When perfused over the blister base, SP induced both plasma extravasation and vasodilatation responses. These responses were significantly inhibited in the presence of either opioid receptor agonist in a naloxone reversible manner. DSLET inhibited SP responses in a dose dependent manner and was 100 times more potent than DAGO. The role of primary afferent sensory nerve terminals in these modulatory effects was investigated in rats pretreated as neonates with capsaicin. The ability of DAGO and DSLET to inhibit the inflammatory response in these rats was significantly less than that in controls. The data raises the possibility that the inhibitory effect of the opioid receptor agonists on the inflammatory response might reflect a role for opioids in modulating tachyphylaxis to SP.

Substance P induced hydrolysis of inositol phospholipids in rat skin in an in vivo model of inflammation.

The present study was undertaken to study the ability of substance P (SP) to induce inositol phospholipid (IP) hydrolysis measured as inositol mono-phosphate (IP1) accumulation, in an in vivo blister model of neurogenic inflammation in the rat hind footpad. SP was found to induce IP1 accumulation in a concentration dependent manner. The use of SP analogues (SP5-11 and SP1-7) indicated that the response is mainly mediated by the C-terminal sequence of the peptide. The response was significantly reduced by the SP antagonist spantide, suggesting that the response is mostly due to activation of the SP receptor on small diameter vessels. Capsaicin pretreatment did not have an effect on the ability of SP to induce the response. Experiments with mepyramine suggest that the response is also partly mediated by SP induced histamine release from mast cells. This is the first study to provide direct evidence for phosphoinositide mediated SP effects in the skin.

Sequence of events in substance P-mediated plasma extravasation in rat skin.

Using a blister model of inflammation in the rat hind footpad, we have studied the temporal and quantitative contribution of mast cell mediators and prostaglandins to substance P-induced plasma extravasation. In addition substance P-related peptides (neurokinin A, SP5-11 and SP1-7) were tested for their ability to induce a plasma extravasation response and the extent of histamine involvement to the response was determined. The present results show that the plasma extravasation response to substance P consists of an early substance P-mediated response that is independent of other mediators and a late response that involves interaction between substance P, mast cell mediators and prostaglandins. An early histamine-independent response was also mediated by neurokinin A, a tachykinin that shares a common C-terminal with substance P and by a C-terminally directed analogue of substance P, namely SP5-11. On the other hand, a late histamine-dependent response was mediated by the N-terminally directed analogue, SP1-7. The present data are suggestive of a possible sequence of events that might occur during an inflammatory response to substance P and might involve independent actions of its C- and N-terminal.

The quantitative contribution of nitric oxide and sensory nerves to bradykinin-induced inflammation in rat skin microvasculature.

Using a blister model in the rat hind footpad, the present study undertook to examine the relative contribution of sensory nerves and nitric oxide (NO) to the inflammatory response induced by bradykinin (BK). Using this model, combined with laser Doppler flowmetry, we were able to simultaneously monitor two parameters of the inflammatory response, namely vasodilatation (VD) and plasma extravasation (PE). Perfusion of BK (1, 10 or 100 microM) over the blister base elicited both VD and PE responses which were dose-dependent. The VD response was of rapid onset, sustained at the lowest concentration (1 microM), and showed tachyphylaxis at the highest two concentrations (10 and 100 microM). The PE response, however, was delayed in onset at the lower concentration but the response was maintained at all concentrations. The endothelium-independent vasodilator, sodium nitroprusside. (SNP, 100 microM), was used as an internal control and elicited a rapid maintained VD response. In rats pretreated as neonates with capsaicin to destroy primary sensory afferents, the inflammatory response to 10 microM BK was significantly smaller (50% and 64% decrease in VD and PE, respectively). The selective inhibitor of NO synthase, NG-nitro-L-arginine (L-NORAG) at 100 microM significantly attenuated the inflammatory response to BK in control rats (76% and 60% decrease in VD and PE, respectively) with a further decrease in the response in capsaicin pretreated rats. The inactive stereoisomer NG-nitro-D-arginine (D-NORAG) (100 microM) did not affect the inflammatory response to BK. The vasodilator response to SNP was intact in capsaicin pretreated rats and was not affected by either L-NORAG or D-NORAG.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin modulates substance P-induced plasma extravasation and vasodilatation in rat skin by an action through capsaicin-sensitive primary afferent nerves.

Using a blister model of inflammation in the rat hind footpad, the present study was undertaken to examine the ability of serotonin (5-HT) to modulate an inflammatory reaction manifested as plasma extravasation and vasodilatation induced by the neuropeptide substance P (SP). In addition, the role of primary afferent sensory nerve fibres in these modulatory effects was studied in capsaicin pretreated rats. Using a protocol of simultaneous perfusion of amine and peptide over the blister base, no major modulatory effect was observed. On the other hand, using a protocol of sequential perfusion, 5-HT was found to extend the plasma extravasation and vasodilatation responses to SP. 5-HT maintained the plasma extravasation response to SP after cessation of stimulation (during the post-stimulation period). On the other hand, it extended the vasodilatation response to SP during the actual stimulation period by preventing the occurrence of tachyphylaxis. These modulatory effects were absent in capsacin-pretreated rats. The present study provides evidence for the first time in vivo to suggest that serotonin can modulate an inflammatory response to SP via a mechanism that involves capsaicin-sensitive sensory fibres.

Release of substance P from peripheral nerve terminals following electrical stimulation of the sciatic nerve.

Substance P is a putative mediator of neurogenic inflammation, where it is postulated to be released from nerve terminals in the skin in response to noxious and electrical stimulation. To demonstrate release of substance P from cutaneous nerve terminals a blister model has been established. Electrical stimulation of the sciatic nerve for 20 min at 50 V, 15 Hz, 0.5 ms, resulted in a significant increase in substance P-like immunoreactivity (SPLI) in blister fluid, which was abolished after neonatal pretreatment of rats with capsaicin. There was no increase of SPLI, following stimulation of the sciatic nerve at 4 V. There was a corresponding decrease in skin SPLI following nerve stimulation at 50 V. Characterization of blister fluid SPLI after stimulation at 50 V demonstrated a peak of immunoreactivity, which co-eluted with synthetic substance P and SPLI from rat skin. These results confirm the hypothesis that electrical stimulation results in release of substance P from nerve terminals in the skin, and therefore fulfils an important criterion of its role as a mediator of neurogenic inflammation.

Mechanisms underlying the vascular activity of beta-amyloid protein fragment (beta A(4)25-35) at the level of skin microvasculature.

Deposition of beta-amyloid protein (beta A4) in extracellular senile plaques is a pathologic hallmark of Alzheimer's disease (AD). The neurotoxic effect of beta A4 has been ascribed to a discrete 11-amino acid internal sequence (beta A(4)25-35). Substance P (SP) has been found to be depleted in the brain of AD patients while its presence was found to protect against the neurodegenerative effect of beta A(4)25-35. Our previous studies, in vivo, in aged rats showed that beta A(4)25-35 exhibits a potent vasoconstrictor (VC) effect in rat skin microvasculature and can prevent SP but not calcitonin gene-related peptide (CGRP) from inducing a vasodilator (VD) response. It was postulated that beta A(4)25-35 might be interacting with SP at the level of the second messenger system via the phosphoinositide pathway. Using a blister model of inflammation in the rat hind footpad, we examined the ability of beta A(4)25-35 to modulate the vascular activity of bradykinin (BK) and serotonin (5-HT) which also activate the phosphoinositide pathway. In addition, the role of nitric oxide (NO), endothelin (ET, an endothelium-derived constrictor factor) and protein kinase C (PKC) in the vascular effects of beta A(4)25-35 were examined using the NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG), the ET-receptor antagonist, BQ-123, and the PKC inhibitor, bisindolylmaleimide (BIM) respectively. Changes in microvascular blood flow were monitored using laser Doppler flowmetry and the area within the response curve measured. The results showed that beta A(4)25-35 (10 microM) induced a VC effect and inhibited the subsequent VD response to BK (10 microM) and 5-HT (1 microM) in a similar fashion to its effect on SP (1 microM). In the presence of L-NOARG (100 microM), the VD effect of SP was reduced and further attenuated after perfusion of beta A(4)25-35. Superfusion of the blister base with BQ-123 (10 microM) or BIM (1 microM) prior to and during perfusion with beta A(4)25-35 abolished its VC effect and allowed SP to induce a normal VD response in both young and old rats. Based on these results, we suggest that the vascular activity of the active fragment, beta A(4)25-35, is mediated by ET via activation of PKC. This study provides new findings which may help to elucidate the signal transduction mechanisms involved in the vascular activity of beta A(4)25-35. The relevance of these mechanisms to those underlying the pathological effects of beta A4 and their significance in AD remains to be determined.