Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 μM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31±5 to 74±14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10–100 μM), DAGO (1–100 μM), DPLPE (10–100 μM), but not U50488H (100 μM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.     

Differential Effects of Increasing Gestational Age and Placental Restriction on Tyrosine Hydroxylase, Phenylethanolamine N-Methyltransferase, and Proenkephalin A mRNA Levels in the Fetal Sheep Adrenal

Abstract: We have demonstrated that there are differential changes in the levels of tyrosine hydroxylase (TH), phenylethanolamine N -methyltransferase (PNMT), and proenkephalin A (Pro Enk A) mRNA in the fetal sheep adrenal during late gestation. Adrenal TH mRNA:18S rRNA ratios increased between gestational days 100 (0.98 ± 0.13; n = 6) and 125 (1.40 ± 0.15; n = 6) and then decreased, whereas adrenal PNMT mRNA:18S rRNA ratios increased regularly between gestational days 100 (0.08 ± 0.01) and 146 (0.17 ± 0.03). The ratio of adrenal Pro Enk A mRNA to 18S rRNA was higher at gestational day 125 (0.085 ± 0.005) than at either 80–100 days (0.038 ± 0.007) or 140–146 days of gestation (0.055 ± 0.013). In 12 ewes, the growth and development of the placenta were restricted (placental restriction group) from conception. The ratio of adrenal PNMT mRNA to 18S rRNA was significantly reduced in the placental restriction group of fetal sheep (0.003 ± 0.002) compared with controls (0.011 ± 0.002), and there was a significant correlation between the ratio of adrenal PNMT mRNA to 18S rRNA and the mean arterial P o ₂ ( r = 0.88, p < 0.0005). In contrast, TH mRNA and Pro Enk mRNA were unaffected by placental restriction. Adrenaline and nonadrenaline syntheses are therefore differentially regulated in the adrenal during late gestation and in response to chronic intrauterine hypoxemia.     

Cardiovascular and thermoregulatory responses to repeated anticholinesterase administration

1. 1.|Pyridostigmine administration decreased resting heart rate by 11 ± 7 beats/min and resting oesophageal temperature by 0.23 ± 12°C after 50 h (P < 0.05). In addition, red blood cell cholinesterase activity was decreased an average of 43 ± 7% after 50 h of pyridostigmine treatment.

2. 2.|The lower heart rates and core temperatures at rest were continued during high intensity exercise in a 35°C environment. Whole body sweating was 12 ± 18% higher (P = 0.20) during exercise in the heat after 50 h of pyridostigmine treatment.

3. 3.|Repeated anticholinesterase administration had little effect on cardiovascular and thermoregulatory responses during high intensity exercise.

Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na, K-ATPase

The rotational motion of an ouabain spin label with sheep kidney Na,K-ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) measurements. Spin-labelled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 ± 0.1 mol of bound ouabain spin label per ATPase β dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (> 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST-EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 ± 11 μs at 0°C and 44 ± 4 μs at 20°C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements with glutaraldehyde-crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K-ATPase-containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K-ATPase for a variety of NMR and EPR investigations.     

Plasma and scales levels of interleukin 18 in comparison with other possible clinical and laboratory biomarkers of psoriasis activity

The aim of the study was to assess plasma and scales levels of interleukin (IL) 18 collected from psoriatic patients with different disease activity. IL-18 concentrations were measured using an enzyme immunoassay in the plasma and scales of 34 patients with chronic plaque type psoriasis. IL-18 levels were analysed with respect to plasma-transforming growth factor β₁ (TGF-β₁), the disease duration and the duration of the present relapse, and psoriasis area and severity index (PASI). Plasma IL-18 concentration varied from 90 to 1300 pg ml^-1 and means (368.2±42.4 pg ml^-1) were significantly elevated in comparison with healthy controls (205.9±31.8 pg ml^-1). The presence of IL-18 was also demonstrated in scales from skin lesions. Treatment caused a significant decrease of plasma IL-18 concentration to 250.2±13.8 pg ml^-1. There was a significant correlation between plasma IL-18 levels and PASI values (r=0.554). There was no correlation between IL-18 concentration in scales and PASI, between IL-18 concentrations in plasma and scales, and between plasma IL-18 and the disease duration or duration of present relapse. Plasma TGF-β₁ concentration demonstrated a significant correlation with PASI (r=0.353), but not with IL-18 levels in plasma (r=0.063) and scales (0.141). The sum of plasma levels of IL-18 and TGF-β₁ divided by the optimal coefficient demonstrated a statistically significant correlation with the highest r-value. The findings confirm an association between plasma IL-18 concentration and psoriasis severity. Moreover, it was shown that combined measurement of IL-18 and TGF-β₁ in plasma can be considered as a possible biomarker of psoriasis activity.     

Improved method for the routine analysis of acetylcholine release in vivo: quantitation in the presence and absence of esterase inhibitor

An improved high-performance liquid chromatographic (HPLC) method using electrochemical detection (ED) is described capable of routinely measuring the low levels of acetylcholine (ACh) typically found in rat brain microdialysis samples. Microdialysis was performed in the striatum of the urethane anesthetized rat using a 4-mm membrane length, high recovery (40% at 1.0 μl/min; ambient conditions), loop-design probe perfused with an artificial cerebrospinal fluid (aCSF) solution containing physiologically normal calcium levels (1.2 mM). The HPLC method utilizes a polymeric stationary phase to resolve choline (Ch) from ACh. These analytes are then converted to hydrogen peroxide (H₂O₂) by a solid-phase reactor (containing immobilized choline oxidase and acetylcholinesterase enzymes). The H₂O₂ is detected amperometrically and quantitated on a platinum (Pt) working electrode (+300 mV; with a unique analytical cell featuring a solid-state palladium reference electrode). Two designs of the Pt working electrode were examined, differing only in the support material used (Kel-F or PEEK). The Kel-F/Pt electrode had a limit of detection (LOD) for both analytes of <30 fmol per 10 μl with a signal-to-noise ratio of 3:1. Striatal microdialysis perfusates were monitored for ACh and Ch over a 0–1000 nM range of neostigmine (NEO) in the CSF perfusion medium. Using the 4-mm probe, basal ACh and Ch levels were detected with a NEO level as low as 10 nM and were found to be 37 ± 3 fmol and 22 ± 1 pmol per 10 μl (mean ± S.E.M., n = 6 replicates) respectively. In similar experiments using 3-mm concentric probes comparable (lower) levels of ACh were found with the 50 and 1000 nM NEO doses (n = 4–21 animals). ACh could not be reliably quantitated when animals were perfused with the 10 nM dose of NEO (n = 4). The PEEK/Pt electrode had an improved LOD of < 20 fmol per 10 μl due to a two- to three-fold decrease in the background noise component. Basal striatal levels of ACh in the absence of NEO approached the LOD and were found to be 15 ± 2 fmol per 10 μl; Ch was 5 ± 1 pmol per 10 μl (n = 2, mean of five basal samples). The analytical system requires very little maintenance; a simple electrochemical electrode cleaning step eliminates the need for routine polishing of the Pt electrode and the mobile phase is stable for up to one week. Both ACh and Ch are resolved in under 7 min making this method highly suitable for analysis of microdialysis samples.     

Effect of histamine and cimetidine on amino acid meal-stimulated gastrin release at a controlled intragastric pH in healthy human beings

Results of several experiments have suggested that histamine-2 receptors play an inhibitory role in regulating gastrin release. We evaluated this prospectively in healthy human beings by infusing intravenously either histamine (0.33 μg/kg/min) or cimetidine (3.33 mg/min) during a continuous 3-h intragastric infusion of a 3% mixed amino acid meal, a potent stimulus of gastrin release. In order to be certain that effects of histamine or cimetidine on gastrin release were independent of their known effects on gastric acid secretion, intragastric pH was maintained at 5.0 by in vivo intragastric titration with sodium bicarbonate or hydrochloric acid. Although histamine and cimetidine had significant effects on gastric acid secretion, neither significantly affected the rises in serum gastrin concentrations during intragastric amino acid infusion. For example, mean gastrin rises above basal concentrations were 39 ± 9 pg/ml on the control day, 39 ± 9 pg/ml on the histamine day and 44 ± 11 pg/ml on the cimetidine day (P > 0.05). Thus, blockade or stimulation of H₂-receptors at the doses tested had no effect on gastrin release in response to an amino acid meal in humans when intragastric pH was maintained at 5.0.     

Effect of hypoxia and glucose deprivation on ATP level, adenylate energy charge and [Ca]0-dependent and independent release of [H]dopamine in rat striatal slices

Release of [³H]dopamine ([³H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [³H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca²⁺]₀-independent release of [³H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 ± 4.15 to 6.98 ± 1.83 mol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C. under glucose-free conditions was significantly reduced from 0.73 ± 0.04 to 0.44 ± 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 ± 0.11 mol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 ± 0.37 nmol/mg protein. The resting release was enhanced in a [Ca²⁺]₀-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 μM ouabain, inhibitor of Na⁺/K⁺-activated ATPase, enhanced the release of [³H]DA at rest in a [Ca²⁺]₀-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na⁺/K⁺-ATPase that results in reversal of the uptake processes and in [Ca²⁺]₀-independent [³H]DA release from the axon terminals.     

Decreased dynorphin levels and increased kappa opioid receptor binding in male rats with isolation-induced hypertension

The role of hippocampal dynorphin A (1–8) (Dyn A (1–8)) and kappa opioid receptors was investigated in the isolation-induced hypertensive rat (IHR). Male Sprague Dawley rats were either isolated (1 per cage) or grouped (3 per cage) for 7 days. Isolated rats exhibited increased blood pressure (systolic blood pressure 159.7 ± 6.6 mmHg) whereas the grouped rats remained normotensive (systolic blood pressure 137 ± 6.3 mmHg). Using radioligand binding techniques we observed a significant increase in kappa opioid receptor binding in the hippocampus of isolated rats (56% increase) and this further increased when the length of isolation was increased to 2 weeks (72% increase). Radioimmunoassay showed that isolation decreased the hippocampal content of Dyn A (1–8) from 12.7 ± 0.4 to 11.6 ± 0.2 pg Dyn A (1–8) per 10 mg tissue (rats weighing approximately 100 g) and from 13.3 ± 0.8 to 9.7 ± 1 pg Dyn A (1–8) per 10 mg tissue (approximately 200 g rats). These data suggest that functional alterations in the hippocampal dynorphin system may be involved in the maintenance of isolation induced hypertension.     

Lack of evidence of an interaction between leu-enkephalin and muscarinic-like responses in the frog semicircular canal

Using multiunit recording of action potentials from the whole vestibular nerve, we studied whether opioid peptide leu-enkephalin (Enk) may modulate muscarinic-like responses in semicircular organs of the frog. When acetylcholine (ACh) (0.1-1 mM) was applied with 1 nM Enk, the maximal frequency increase induced by ACh was reduced.However,the frequency decrease of ACh responses under Enk did not differ from the frequency decline in basal spike discharge induced by Enk alone. Administration of atropine (1 microM) left the response to Enk intact and blocked the excitatory effect of ACh. No modification of the ACh response under Enk was observed in the presence of the non-selective opioid receptor antagonist naloxone (10 microM). This study suggests that no interaction exists between the ACh-mediated excitatory action on resting activity in the isolated semicircular canal preparation and the suppressive action of Enk.     

Role of dexamethasone on vasopressin release during endotoxemic shock

The present study was designed to assess the hypothesis that dexamethasone (DEX) through the control of nitric oxide (NO) synthesis could regulate the release of vasopressin (AVP), which plays an important role in the regulation of arterial pressure and plasma osmolality. Endotoxemic shock was induced by intravenous (i.v.) injection of 1.5 mg/kg lipopolisaccharide (LPS) in male Wistar rats weighing 250–300 g. After LPS administration, a group of animals were treated with DEX (1.0 mg/kg of body weight), whereas saline-injected rats served as controls. The LPS administration induced a significant decrease in mean arterial pressure (MAP) with a concomitant increase in heart rate (HR) (ΔVMAP: − 16.1 ± 4.2 mm Hg; ΔVHR: 47.3 ± 8.1 bpm). An increase in plasma AVP concentration occurred and was present for 2 h after LPS administration (11.1 ± 0.9 pg/mL) returning close to basal levels thereafter and remaining unchanged until the end of the experiment. When LPS was combined with i.v. administration of a low dose of DEX, we observed an attenuation in the drop of MAP (ΔVMAP: − 2.2 ± 1.9 mm Hg) and a decrease in NO plasma concentration [NO] after LPS administration (1098.1 ± 68.1 µM) compared to [NO] after DEX administration (523.4 ± 75.2 µM). However, this attenuation in the drop of MAP was accompanied by a decrease in AVP plasma concentration (3.7 ± 0.4 pg/mL). These data suggest that AVP does not participate in the recovery of MAP when DEX is administered in this endotoxemic shock model.     

The role of estrogen in bone growth and maturation during childhood and adolescence

Twenty years ago it was believed that pubertal growth was stimulated by testicular androgen in boys and by adrenal androgen in girls. Estrogen, which was used to inhibit growth in excessively tall girls, was not thought to have growth-promoting effects. We hypothesized that estrogen has a biphasic effect on epiphyseal growth, with maximal stimulation at low levels. We showed that the administration of low doses of estrogen, corresponding to a serum estradiol level of about 4 pg/ml (15 pmol/l) caused more than a 60% increase over the prepubertal growth rate in both boys and girls. To test the hypothesis that estrogen is the principal mediator of the pubertal growth spurt in boys, we administered the aromatase inhibitor, testolactone, to boys with familial male-limited precocious puberty. Testolactone produced near normalization of both growth velocity and bone maturation, despite levels of serum testosterone that remained within the adult male range. The observation that low levels of estrogen stimulate growth and bone maturation suggested that estrogen might explain the more rapid epiphyseal maturation of prepubertal girls compared to boys. To determine whether prepubertal girls have higher estrogen levels than prepubertal boys, we developed an ultrasensitive recombinant cell bioassay for estrogen with a sensitivity of 0.02 pg/ml (0.07 pmol/l) estradiol equivalents. Prepubertal girls had approximately eight-fold higher levels of serum estradiol than did prepubertal boys (0.6 ± 0.6 pg/ml (SD) (2.2 ± 2.2 pmol/l) vs 0.08 ± 0.2 pg/ml (0.29 ± 0.73 pmol/l), P < 0.05). We concluded that the pubertal growth spurt of both sexes is driven primarily by estrogen, and that the more rapid epiphyseal maturation of prepubertal girls (vs boys) may be explained by their higher estradiol levels.     

Endothelin-1 binding sites and inositol-monophosphate formation in kidney medulla from adult and juvenile SHR and WKY rats

High resolution autoradiography at the cellular level localized endothelin-1 binding sites to the collecting ducts in the rat renal papilla. These receptors were functionally coupled to the phosphatidyl-inositol system since endothelin-1 stimulated the accumulation of IP₁ in a concentration-dependent manner in cross chopped slices from renal papillae. The concentration-effect curves in 4 week old normotensive Wistar-Kyoto rats (WKY) lay to the right of curves from 4 week old and adult spontaneously hypertensive rats (SHR) and adult WKY (20–24 week old) animals; the EC₅₀ values in the 4 week old animals were 17 ± 5 and 8 ± 1 nM (P < 0.05, N = 5; mean ± SE) for the normotensive and hypertensive animals, respectively. Autoradiographic studies showed that the density and distribution of binding sites for [¹²⁵I]endothelin-1 in the kidneys did not differ between the groups; receptor densities in the renal papillae were 461 ± 37 (fmol/mg protein) in the 4 week old WKY, and 443 ± 27 (fmol/mg protein) in the 4 week old SHR. The plasma levels of immunoreactive endothelin-1 were also similar between groups; 4 week old SHR (39 ± 3 pg/ml) and 4 week old WKY (36 ± 1 pg/ml). The increased response to endothelin-1 may be related to the development of the hypertensive state in the SHR.     

Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.     

Temperature acclimation of axonal functions in the crayfish Astacus astacus L.

1. 1.|Crayfish (Astacus astacus L.) were acclimated for 1–3 weeks at 5 and 20°C. The effects of temperature on the functions of the unicellular medial giant axon were studied.

2. 2.|The resting membrane potential of the giant axon increased slightly with the experimental temperature from 2 to 32°C. The temperature dependence of the resting membrane potential could be described by two lines, which intersected at about 12°C in cold-acclimated crayfish and at about 16°C in the warm-acclimated.

3. 3.|The amplitude of the action potential was stable at temperatures from 4 to 26°C. It decreased at temperatures above 26°C in both acclimation groups.

4. 4.|The duration of the falling phase of action potential was highly temperature dependent at low temperatures. A break in the slope of the dependence was found at about 14°C in cold-acclimated crayfish and at about 17°C in the warm-acclimated.

CNS thermosensitivity and control of latent heat dissipation in the pigeon

Thermoregulatory responses to heat exposure were studied in 12 hand-reared, acclimated pigeons (Columbia livia). Measurements of body temperature (Tcl), brain temperature (Tbr), cutaneous water evaporation (CWE) and respiratory frequency (fr) were carried out in intact conscious heat exposed birds. In a second group of lightly restrained birds, fr and CWE were taken when temperatures of the trunk, brain and air (Ta) were independently changed. Increasing Tbr to 43.5–43.8°C induced a pronounced polypnea (deep and fast, (300 breaths min⁻¹) when Tcl regulated at 42.4°C. Moreover, when hyperthermia (Tcl = 43.0°C) was combined with increased Tbr (43.0–43.8°C) shallow and fast panting (>500 breaths min⁻¹) was evoked. CWE was probably elicited by inputs generated by the skin warm receptors as a result of increased Ta. Moreover it was demonstrated that warming the brain to 42.5°C elicits cutaneous water evaporation in birds exposed to 26°C. When a high Ta (60°C) is accompanied by a high relative humidity (17%), the combined effect generates inputs eliciting intensive panting. The integration of the present and earlier data allows us to generate a model demonstrating the distinguished significance of the trunk, skin and brain thermosensors in the regulation of both respiratory and cutaneous latent heat dissipation. The present model also emphasizes the fact that the highly thermosensitive pigeon brain responds in a similar pattern to that found in mammals     

Protein size distribution and inhibitory effect of wheat hydrolysates on Neutrase

Neutrase^®, used for hydrolysis of wheat proteins, was inhibited by end-product in a competitive uncompetitive way. The inhibition ratio depends on the progress of protein hydrolysis (degree of hydrolysis) and it remains constant beyond a degree of hydrolysis of 7.5%. The inhibitor was separated, on Sephadex G-25 column, in three fractions (>2.4, 2.4–0.5 and <0.5 kDa) generating an inhibition ratio of 29%, 46% and 67% respectively. The peptides size distribution (<1, 1–2, 2–3 and >3 kDa) of fractions was determined using size exclusion-high performance liquid chromatography. The analysis of obtained data, using a simple mathematical regression, showed a correlation factor of 0.98 between the inhibition ratio and the peptides less than 1 kDa and 0.99 when considering the peptides lower than 1 kDa and higher than 3 kDa.     

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.     

The effect of acclimation on the acid-base status of pigeons exposed to high ambient temperatures

Arterial pH, PCO₂ (P_aCO₂), plasma bicarbonate [HCO₃⁻ and respiratory frequency were measured in pigeons exposed to ambient temperatures (T_aS) of 30–60°C. Acclimated, nonpanting birds regulated acid-base balance at normal levels, when exposed to T_as) between 30 and 53°C T_a. At higher T_as (55–60°C), both nonpanting and panting acclimated pigeons regulated pH at normal levels, 7.544 ± 0.011 (SD) and 7.531 ± 0.022 (SD), respectively, accompanied by a slight hypocapnia, 24.8 ± 4.0 Torr and 23.8 ± 2.49 Torr (P_aCO₂), respectively. Nonacclimated birds, exposed to 50°C T_a, endured a severe hypocapnia (P_aCO₂ of 9.1 ± 2.52 Torr) and alkalosis (pH of 7.702 ± 0.048). Thirteen exposures to > 50°C T_a, 4–6 h a day, resulted in a significant improvement in the capacity of the panting pigeon to maintain an almost normal acid-base balance, i.e. actual and standard [HCO₃⁻ of 22.6 ± 1.22 and 25.7 ± 1.10 mM/l, respectively, and only a slight hypocapnia (P_aCO₂ of 23.6 ± 3.9 Torr) and alkalosis (pH of 7.589). The suggestion that acclimation to high T_as (50–60°C) is needed for fine adjustment between the competing needs for heat dissipation, pulmonary gas exchange, and acid-base regulation in the heat-exposed pigeon is discussed.     

Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa

Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS–PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 μg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29 ± 2.7, 2.61 and 0.2 mg/ml, respectively. Eighteen total protein bands were observed in the range of 12–127 kDa. Eight major HB proteins were isolated in the range of 13–71 kDa. Seven major GB proteins were isolated in the range of 13–61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9 ± 0.6) followed by GB proteins (25.4 ± 0.6) and control (21.2 ± 1.4). The difference in BCMPT values between protein treated and control group was significant (P < 0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4 ± 0.65 and 66.1 ± 0.6, respectively). The difference in HOST values between proteins treated and control group (50.4 ± 2.0) was significant (P < 0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin–sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 μg/ml gave best results) of buffalo cauda spermatozoa.