Maria A. Matuszek

Pharmacological studies of jumper ant (Myrmecia pilosula) venom: evidence for the presence of histamine, and haemolytic and eicosanoid-releasing factors.

Jumper ant venom was prepared by extraction of venom sacs in distilled water and centrifugation to remove insoluble material. Jumper ant venom (2 micrograms/ml) produced a biphasic response on isolated guinea-pig ileum, i.e. an initial rapid contraction followed by a slower prolonged contraction. The histamine antagonist mepyramine (0.1 microM) inhibited the first phase of this response by greater than 90%. In the isolated rat stomach fundus strip (which is insensitive to histamine), jumper ant venom (6 micrograms/ml) produced only a single contraction. No tachyphylaxis was observed to repeated doses of jumper ant venom in guinea-pig ileum or rat fundus strip. Responses to jumper ant venom of the egg-albumin-sensitised guinea-pig ileum were not significantly different before and after an in vitro anaphylactic response induced by egg albumin (0.5 mg/ml). Fluorometric assay revealed a mean value of 0.9 +/- 0.2% of the dry weight as histamine in jumper ant venom. Both the lipoxygenase/cyclo-oxygenase inhibitor BW755C and the cyclooxygenase inhibitor indomethacin significantly inhibited the second phase response to jumper ant venom of the guinea-pig ileum, and the response of the rat fundus strip. The muscarinic receptor antagonist atropine (0.1 microM), the bradykinin antagonist [Thi5,8,D-Phe7]-bradykinin (10 microM) and the angiotensin converting enzyme inhibitor captopril (20 microM) did not affect either phase of the venom response in guinea-pig ileum. Jumper ant venom caused haemolysis of guinea-pig blood. The degree of haemolysis was significantly reduced when boiled venom was used. These results suggest that jumper ant venom contains histamine and may cause the release of cyclo-oxygenase products. It also contains a heat-sensitive haemolytic factor.

Some enzymic activities of two Australian ant venoms: A jumper ant Myrmecia pilosula and a bulldog ant Myrmecia pyriformis

Venoms from two related Australian ants, a jumper ant (Myrmecia pilosula) and a bulldog ant (Myrmecia pyriformis), were quantitatively analysed for the following enzymic activities: phospholipase A2, phospholipase B, phospholipase C, hyaluronidase, esterase, acid phosphatase, alkaline phosphatase and phosphodiesterase. Both venoms contained phospholipase A2, phospholipase B, hyaluronidase, acid phosphatase and alkaline phosphatase activities. Myrmecia pyriformis venom had significantly greater phospholipase B, acid phosphatase and alkaline phosphatase activities than Myrmecia pilosula venom. No detectable quantities of phospholipase C, esterase or phosphodiesterase activities were found in either venom.

Haptoglobin elutes from human atherosclerotic coronary arteries—a potential marker of arterial pathology

BACKGROUND Molecules which egress from atherosclerotic arteries may function as plasma markers of arterial pathology, but such egress has not been proven with living human coronary arteries. We hypothesised that proteins eluting from the arterial wall may discriminate between atherosclerotic and non-atherosclerotic coronary arteries. METHODS AND RESULTS During cardiac bypass surgery, 155 sequential fractions of antegradely flushed coronary cardioplegia solution were collected by balloon-cuffed catheter from the coronary sinus in subjects with angiographically extensive (n=30) or minor (n=7) coronary disease. Although plasma was the major source of protein in heavily blood-contaminated samples, under conditions of low blood contamination (<0.5 mg/ml red cell Haemoglobin) coronary circulation-derived protein was detected. N-terminal sequencing of a major 40 kDa band detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated 100% homology with beta chain of Haptoglobin (Hpt). Comparison of perfusates from patients with and without significant coronary disease found that the concentration of Hpt was markedly increased in perfusates from atherosclerotic coronary arteries (0.099+/-0.017 microg Hpt/microg Hb) relative to controls (0.016+/-0.008 microg Hpt/microg Hb, P=0.0027). Analysis of peripheral plasma samples of the same subjects, and of a separate cohort of patients, confirmed greater Hpt in those with angiographic coronary disease than in those without disease. CONCLUSIONS Proteins such as Hpt elute from the human coronary vascular bed and may differentiate between arteries with minor or extensive atherosclerosis. Although the suitability of Hpt as a circulating plasma marker for atherosclerosis remains to be established, the approach used in the present study may permit identification of diverse plasma-detectable markers of atherosclerosis, and the subsequent non-invasive evaluation of in vivo arterial pathology.

An investigation of tachykinin NK2 receptor subtypes in the rat.

The heterogeneity of tachykinin NK2 receptor subtypes was examined in five tissues from the rat, using binding and functional techniques. Initial experiments with the selective radioligand [125I][Lys5,Tyr(I2)7,MeLeu9,Nle10]neurokinin A-(4-10) showed no specific binding to rat spinal cord membranes or sections. However, this radioligand exhibited high specific binding (80-95% of total) in membranes from the rat fundus, colon, bladder and vas deferens. Dissociation constants (KD) were lower in bladder and colon (0.4 nM) than in fundus (1.9 nM) or vas deferens (1.4 nM). Neurokinin A, neuropeptide gamma, [Lys5,MeLeu9,Nle10]NK(4-10), SR 48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophen yl)butyl]benzamine], GR 94800 [PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-Nle-NH2] and MEN 10627 [cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)] displayed high affinity (pIC50 8.4-9.5) as competitors, with no significant difference in potency between these four tissues. [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) contracted the isolated fundus (EC50 117 nM) and bladder (EC50 10 nM) and these responses were similarly inhibited by the tachykinin NK2 receptor antagonists, SR 48968 and MEN 10627 (pA2 values 7.6-8.2). In spite of differences in KD seen in some tissues, these results do not provide compelling evidence for tachykinin NK2 receptor heterogeneity in smooth muscle-containing tissues in the rat. The absence of detectable binding in rat spinal cord may be due to very low expression of tachykinin NK2 receptors, or to existence of a different receptor subtype.

Binding and functional potency of neurokinin A analogues in the rat fundus: A structure-activity study.

Structure-activity relationships of neurokinin A (NKA) and the two analogues NKA(4-10) and [Nle¹⁰]NKA(4-10) were investigated at the rat fundus NK-2 receptor, using selected amino acid substitutions. Both radioligand binding with [¹²⁵I][Lys⁵,Tyr(I₂)⁷,MeLeu⁹, Nle¹⁰] NKA(4-10) and functional studies were performed and correlated. In membrane binding experiments loss of His¹ and Lys², or replacement of Lys² with Ala did not substantially alter binding affinity of NKA. NKA(4-10) free acid was unable to compete with the radioligand. [Nle¹⁰]NKA(4-10) binding affinity to rat fundus membrane preparations was decreased when substituting Asp⁴ with Gln or Asn, or Val⁷ with either Tyr or Ile. Replacement of Ser⁵ with the negatively charged Glu also decreased the binding affinity, but substitution with the positively charged Lys substantially increased the affinity of [Nle¹⁰] NKA(4-10) for the NK-2 receptor. Lengthening NKA(4-10) or [Nle¹⁰]NKA(4-10) with Ala¹¹ or Nle¹¹, respectively, decreased the binding affinity of the peptide. In both binding and functional studies, replacement of any of the residues of NKA(4-10), except for Ser⁵, with alanine decreased the affinity of the peptide for the NK-2 receptor. Ala substitutions at positions 4, 6, and very obviously at 8, 9 and 10 of NKA(4-10) yielded peptides unable to achieve a maximum contractile response, although they did not demonstrate antagonist activity. These data confirm the importance of the NKA carboxyl terminus, and the requirement for Phe⁶, Val⁷, Gly⁸, Leu⁹ and Met¹⁰ integrity for interaction with the NK-2 receptor. They also suggest that Ser⁵ is a good site to target modifications leading to the design of new potential drugs.

Smooth Muscle, Neurons and Interstitial Cells of Guinea Pig Ileum: Are There Tachykinin Neurokinin 1 Receptor Subtypes?

Binding and autoradiographic studies were carried out in guinea pig ileum to examine neurokinin (NK) 1 receptor location and subtypes using the NK-1-selective radioligand [125I]Bolton-Hunter[Sar9,Met(O2)11]SP. Two membrane preparations were made: (1) longitudinal muscle containing the myenteric plexus and (2) circular muscle containing the interstitial cells of Cajal. In saturation binding studies, the KD was estimated as 1.3 and 1.0 nmol/l in each preparation, respectively. In competition binding, the rank order of potency was similar in both membrane preparations: SR140333 ≈ CP99994 ≧ [Sar9,Met(O2)11]SP ≈ physalaemin ≈ CP96345 (pIC50 9.5–8.7) >> neuropeptide γ ≧ septide (pIC50 7.8–7.4). Similarly, scyliorhinin I displayed equal affinity in both preparations, although binding was at two sites, of high affinity (pIC50 9.1, 30%) and low affinity (pIC50 7.2–6.6, 70%). The only competitor to bind differently in the two muscle preparations was scyliorhinin II, which bound to one site with low potency in the circular muscle (pIC50 6.9) but to high-affinity (pIC50 9.0, 17%) and low-affinity (pIC50 6.7, 83%) sites in the longitudinal muscle. In autoradiographic studies, dense specific binding was associated with the myenteric plexus and the inner circular muscle containing the interstitial cells of Cajal, with minimal specific binding to longitudinal and circular smooth muscle. These results suggest that the NK-1 receptor on the interstitial cells and the myenteric plexus is similar. The apparently low numbers of binding sites on intestinal smooth muscle may be due to a low expression of the NK-1 receptor. Alternatively, the radioligand may not recognize the guinea pig ileum muscle NK-1 receptors due to possible minor differences in their sequence or glycosylation.

Increased Insulin following an Oral Glucose Load, Genetic Variation near the Melatonin Receptor MTNR1B, but No Biochemical Evidence of Endothelial Dysfunction in Young Asian Men and Women.

Aim To identify biochemical and genetic variation relating to increased risk of developing type 2 diabetes mellitus and cardiovascular disease in young, lean male and female adults of different ethnicities. Method Fasting blood and urine and non-fasting blood following oral glucose intake were analysed in 90 Caucasians, South Asians and South East/East Asians. Results There were no differences in age, birthweight, blood pressure, body mass index, percent body fat, total energy, percentage of macronutrient intake, microalbumin, leptin, cortisol, adrenocorticotropic hormone, nitric oxide metabolites, C-reactive protein, homocysteine, tumor necrosis factor-α, interleukin-6, von Willebrand factor, vascular cell adhesion molecule-1, plasminogen activator inhibitor-1, and tissue plasminogen activator. Fasting total cholesterol (P = .000), triglycerides (P = .050), low density lipoprotein (P = .009) and non-fasting blood glucose (15 min) (P = .024) were elevated in South Asians compared with Caucasians, but there was no significant difference in glucose area under curve (AUC). Non-fasting insulin in South Asians (15–120 min), in South East/East Asians (60–120 min), and insulin AUC in South Asians and South East/East Asians, were elevated compared with Caucasians (P≤0.006). The molar ratio of C-peptide AUC/Insulin AUC (P = .045) and adiponectin (P = .037) were lower in South Asians compared with Caucasians. A significant difference in allele frequency distributions in Caucasians and South Asians was found for rs2166706 (P = 0.022) and rs10830963 (P = 0.009), which are both near the melatonin receptor MTNR1B. Conclusions Elevated non-fasting insulin exists in young South Asians of normal fasting glucose and insulin. Hepatic clearance of insulin may be reduced in South Asians. No current biochemical evidence exists of endothelial dysfunction at this stage of development. MTNR1B signalling may be a useful therapeutic target in Asian populations in the prevention of type 2 diabetes mellitus.

Elevated levels of circulating cortisol in young normotensive adult men with a family history of hypertension.

1 Differences in blood lipids, glucose, insulin, amylin, adrenocorticotropic hormone (ACTH), cortisol, aldosterone, angiotensin II, metabolites of nitric oxide (nitrate, nitrite), asymmetric dimethyl arginine, endothelial leucocyte adhesion molecule‐1, vascular cell adhesion molecule‐1, C‐reactive protein, homocysteine and oxidative status (urate, vitamin A, vitamin E, b‐carotene and total anti‐oxidant capacity) were investigated in men (aged 18–25 years) with (+) or without (–) a family history (FH) of hypertension. 2 In the present study, FH+ was defined as having at least one parent or grandparent taking medication for hypertension. Blood (60 mL) was sampled (0800–1000 hours) from a cannulated forearm vein after an overnight fast and 24 h abstinence from caffeine‐containing products and alcohol. 3 Comparing FH+ with FH–, systolic blood pressure (124 ± 1 vs 117 + 3 mmHg, respectively; n = 50 and 14, respectively; P < 0.05) and plasma cortisol (377 ± 23 vs 298 ± 24 nmol/L, respectively; n = 43 and 12, respectively; P < 0.05) were found to be significantly higher in the former group. 4 No significant difference was found between the two groups for body mass index, resting heart rate, diastolic and mean blood pressures or any of the biochemical measures studied. 5 A significant correlation was found between cortisol and ACTH (r = 0.73). No correlation was found between cortisol and any other parameter measured. 6 These data indicate that elevated cortisol levels are characteristic of young lean normotensive FH+ men. The future impact of this on their vascular health and hypertension remains to be determined.

Impact of cortisol on α-actin content in vascular smooth muscle cells of fetal sheep

1 The effects of gestation on a‐actin levels in vascular smooth muscle aortae were studied in 31 fetal sheep, aged 66–144 days (term = 150 days). Aortae were collected post‐mortem. 2 Aortae, carotid and femoral arteries from two groups of chronically catheterized fetal sheep (110–114 days) were also examined. One group was infused with cortisol (n = 6; hydrocortisone sodium succinate, total dose 16.8 mg in 48 h) and the control group received saline (0.15 mol/L, 0.33 mL/h, n = 7). 3 Vascular homogenate protein was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western transfer. a‐Actin was identified using a monoclonal mouse anti‐a actin antibody and standardized against tissue protein and DNA content. 4 Between 60 and 144 days gestation, there was an exponential increase in the a‐actin content of vascular smooth muscle cells from fetal sheep aorta (P < 0.0001). a‐Actin concentration (densitometry units (U) relative to DNA 260 nm absorbance (Abs)) was significantly (P < 0.05) higher in the aortae of cortisol‐infused (12 601 2499 U/Abs) fetal sheep compared with those that were saline‐infused (4514 670 U/Abs). a‐Actin (relative to DNA absorbance) of carotid and femoral vessels in cortisol‐infused animals (20 659 4812 U/Abs) compared with those that were saline‐infused (14 461 2645 U/Abs) was increased, but the difference was not significant. 5 Therefore, the a‐actin concentration of the vascular smooth muscle of the aorta increases throughout gestation. Cortisol treatment is associated with further increases in a‐actin concentration in the fetal aorta, indicating that the development of large conduit vessels can be altered by this glucocorticoid.