Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed-time artificial insemination protocol in Australian Bos indicus cattle.

Increasing use of fixed-time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN2 ) in a Tris-egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post-thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.

Endometrial biopsy in Bos indicus beef heifers.

An endometrial biopsy allows for a comprehensive assessment of the uterine environment of a breeding female. Although routine in mares, devices used for endometrial biopsies are impracticable in heifers due to the size and structure of the cervix. This report describes the use of a human bronchoscopy biopsy device (Karl Storz® 10366L) for collection of endometrial biopsies in Bos indicus beef heifers. The Storz® device is smaller and thinner and enabled the collection of an endometrial biopsy in 86% of heifers (n = 44/51). The biopsied tissue was of good quality and suitable for transcriptomic assessment of the endometrium, with total RNA yield and RNA integrity number (RIN) averaging 1.3 µg (range 0.4-5.3 µg) and 7.4 (range 5.7-8.4), respectively.

Sex and the clinical value of body mass index in patients with clear cell renal cell carcinoma.

BACKGROUND: An increased body mass index (BMI) is significantly associated with favourable prognosis in renal cell carcinoma (RCC). This study investigated the associations among sex, BMI, and prognosis in clear cell RCC patients. METHODS: We retrospectively analysed 435 patients with clear cell RCC who underwent a nephrectomy. The associations among sex, BMI, clinicopathologic factors, and cancer-specific survival (CSS) were analysed. RESULTS: As a continuous variable, increased BMI was associated with higher CSS rate by univariate analysis in the whole population (hazard ratio, 0.888 per kg m(-2); 95% confidence interval, 0.803-0.982; P=0.021). A sub-population analysis by sex demonstrated that BMI was significantly associated with CSS in men (P=0.004) but not in women (P=0.725). Multivariate analysis revealed BMI to be an independent predictor of CSS in only men. CONCLUSION: Body mass index was significantly associated with clear cell RCC prognosis. However, the clinical value of BMI may be different between men and women.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

The long and the short of sperm selection in vitro and in vivo: swim-up techniques select for the longer and faster swimming mammalian sperm.

Sperm competition and sexual selection outcomes are sometimes reported as depending on sperm velocity and flagellar length, suggesting that sperm shape may be optimized for maximum efficiency. This is a largely unexamined assumption regarding sperm performance. Here, we examine this idea using a 'swim-up' selection technique as a proxy for sperm transport within the female tract, testing the hypothesis that variation in sperm tail length should be reduced by this procedure. We detected small but significant (P < 0.001) increases in mean flagellar length in brown hare, pig and bull spermatozoa without reduction in variance. Applying the swim-up technique to boar ejaculates confirmed that the selected populations were enriched for fast motile spermatozoa. These effects were also reflected in vivo where boar spermatozoa with both short and long flagellae were able to reach and colonize the oviductal sperm reservoir. The benefits of possessing a longer flagellum thus appear to be marginal, suggesting that sperm selection in vivo is based on more complex criteria.

Variable sperm size and motility activation in the pipefish, Syngnathus abaster; adaptations to paternal care or environmental plasticity?

Like seahorses, some of the closely-related pipefish species (Family Syngnathidae) incubate their eggs within a male brood pouch. This has contributed to considerable confusion about sperm transfer mechanisms to the eggs; some authors have reported that ejaculates are released directly into water before they reach the eggs, while others have suggested that eggs are fertilised using spermatozoa deposited directly into the brood pouch via an internal sperm duct. Here we present anatomical evidence from the freshwater pipefish, Syngnathus abaster, showing not only that direct sperm deposition into the pouch is impossible, but that spermatozoa must somehow travel a significant distance (>4 mm) outside the body of the male, to reach and fertilise eggs in the pouch. We have also used several putative sperm-activating solutions to identify the type of environment most conducive to sperm activation. Spermatozoa released from the testis were active for a brief period (<5 min) in water or 150 mm saline, but showed prolonged (>25 min) motility in ovarian fluid. This suggests that spermatozoa are released into a mixture of ovarian fluid and eggs while the male and female are in close contact. Our data also suggest that the fertilisation mechanism is highly efficient (sperm : egg ratio <200 : 1) even though this pipefish species produces dimorphic spermatozoa (with long and short flagellae). The shorter (<40 microm) morphotypes were not capable of motility activation, and are therefore probably incapable of fertilisation. If so, the sperm : egg ratio reported here would represent an overestimate.

Morphological defects, sperm DNA integrity, and protamination of bovine spermatozoa.

The association between sperm morphology characteristics and DNA conformation and integrity is still controversial. In bulls, major morphological sperm abnormalities have been associated with reduced fertility, and morphological assessment is used to provide an indication of potential fertility of the individual. Sperm DNA fragmentation and damage has a negative effect on embryo development and subsequently fertility, with bull spermatozoa generally displaying low levels of DNA damage and tight chromatin. However, sensitive methods for detecting chromatin damage may reveal associations with morphological defects. The objective was to determine whether morphological sperm abnormalities and variables expressing sperm DNA integrity and protamination are correlated in bulls, using the sperm chromatin structure assay (SCSA) and the sperm protamine deficiency assay (SPDA). Electroejaculated samples (n = 1009) from two-year-old tropically adapted bulls were split and fixed and submitted to microscopic sperm morphology assessment, and snap-frozen for sperm nuclear integrity assessments by SPDA and SCSA. For SPDA, the variables were defective (MCB) and deprotaminated (HCB), and for SCSA, the variables were DNA fragmentation index (DFI) and high DNA stainability (HDS). HCB correlated with DFI; τKen2  = 0.317 and HDS; 0.098, and MCB correlated with DFI; 0.183 (p < 0.001). The percentage of morphological normal spermatozoa was correlated negatively to DFI; τKen2  = -0.168, MCB; -0.116 and HCB; -0.137 (p < 0.001). HCB and DFI were both positively correlated to head defects, proximal droplets, and spermatogenic immaturity, but not to distal droplets, vacuoles, or diadems. Sperm DNA integrity and protamination, using the SCSA and SPDA, respectively, in bulls show associations with morphological parameters, particularly with head shape abnormalities and indicators of spermatogenic immaturity, including proximal droplets. The vacuoles and diadem defects were not correlated with sperm nuclear integrity, and hence, these are likely physiological features that may not directly affect sperm chromatin configuration.

Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.

Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data.

Isolation and characterization of a rat homologue of the human tuberous sclerosis 1 gene (Tsc1) and analysis of its mutations in rat renal carcinomas.

In the Eker rat, a germ-line mutation in the homologue of the human tuberous sclerosis gene (Tsc2) causes renal cell carcinomas (RCs) with a complete penetrance in all heterozygotes. Tsc2 mutations have also been found in a subset of chemically induced non-Eker rat RCs. Because tuberous sclerosis patients with alteration of either of the two predisposing genes (TSC1 and TSC2) show identical symptoms, the products of these two genes are thought to be involved in a common biological pathway. In this study, to analyze the possible overlap between the functions of Tsc2 and Tscl gene products, we isolated and characterized a rat homologue of the TSC1 gene (Tsc1). The rat Tsc1 gene, which has an identical exon-intron structure to that of human TSC1 and is localized on rat chromosome 3, has been shown to encode a protein (hamartin) that is highly homologous to the human counterpart with an approximately 86% amino acid sequence identity. Using PCR-single-strand conformational polymorphism analysis, we identified two splicing donor site mutations in one chemically induced rat RC (1 of 15). This suggests that alterations of the Tsc1 gene may be involved in the development of a subset of rat RCs.

Amplification of the TCL1 flanking region at 14q32.1 with no TCL1 gene transcription in a patient with peripheral T cell lymphoma.

Cytogenetic and molecular-genetic characteristics in peripheral T cell lymphoma (PTL) have not been well defined, except for those in adult T cell leukemia/lymphoma (ATL/L). Translocations and inversions involving a chromosome band 14q32 were extremely common abnormalities reported in PTL and ATL/L. We studied the involvement of TCL1, a recently isolated gene located in 14q32.1, in tumor tissues from 20 patients with PTL including three with 14q32 translocations by two color fluorescent in situ hybridization (FISH) using two cosmid probes flanking the TCL1 gene. The two cosmid signals were separated in none of them, but much increased in number in one tumor without 14q32 translocation, indicating that the TCL1 genomic region was amplified in this tumor. Reverse transcription-polymerase chain reaction (RT-PCR), however, failed to detect the TCL1 transcript in the tumor. These findings suggest that an oncogene other than TCL1 may be located in 14q32.1, and its amplification may be involved in the neoplastic process of PTL.

Chromosome abnormalities and MLL rearrangements in acute myeloid leukemia of infants.

Of 29 infants with acute myeloid leukemia (AML), 14 (48%) had various 11q23 translocations. MLL rearrangements were examined in 21 of the 29 patients, and 11 (52%) showed the rearrangements. 11q23 translocations and/or MLL rearrangements were found in 17 (58%) of the 29 patients. While all but one of the 17 patients with 11q23/MLL rearrangements had M4 or M5 type of the FAB classification, the 12 patients without such rearrangements had various FAB types, including M2, M4, M4EO, M6 and M7. Of the 12 patients with other chromosome abnormalities or normal karyotypes, two had inv(16) ort(16;16), one had t(1;22)(p13;q13), and two had a novel translocation, t(7;12)(q32;p13). The breakpoint on 12p of the t(7;12) was assigned to intron 1 or the region just upstream of exon 1 of the TEL/ETV6 gene by fluorescence in situ hybridization. The event-free survival at 5 years for the 17 patients with 11q23/MLL rearrangements was 42.2%, and that for the 12 patients without such rearrangements was 31.3% (P = 0.5544). 11q231MLL rearrangements have been frequently reported and a poor prognosis in infant acute lymphoblastic leukemia implied. Our study showed that while 11q23/MLL rearrangements were also common in infant AML, AML infants with such rearrangements had a clinical outcome similar to that of AML infants without such rearrangements.

Validation of an experimental strategy for studying surface-exposed proteins involved in porcine sperm-oviduct contact interactions.

Previous experiments have shown that boar sperm survival in vitro is enhanced when co-incubated with a solubilised protein extract of porcine oviducal apical plasma membrane proteins. Here, we examine the hypothesis that the effects are mediated by direct oviduct-sperm contact and use in situ biotinylation of the oviducal epithelial surface to trace the surface-exposed biotinylated proteins through purification and solubilisation steps. We have also examined the effectiveness of mechanical scraping as a method of recovering oviducal epithelial proteins. We show that a subset of proteins originally exposed at the oviducal surface eventually bind to spermatozoa during incubation in vitro, but also show that a different protein subset is implicated if the sperm incubation is performed with proteins that had been biotinylated after (ex situ) extraction from the oviduct. Apical plasma membrane fractions biotinylated after purification contained many more biotinylated protein bands than preparations labelled before purification and multiple protein bands were eventually found to associate with spermatozoa. Although the evidence presented here supports the hypothesis that protein(s) anchored to the oviducal epithelium bind populations of spermatozoa directly and may have a role in the enhancement of sperm viability, it also shows that the choice of investigative technique exerts a major influence on experimental outcomes.

Electrophysiological effects of FK664, a new cardiotonic agent, on preparations from guinea pig ventricle and from rabbit sino-atrial node.

Effects of the cardiotonic agent FK664, 6-(3, 4-dimethoxy-phenyl)-1-ethyl-4-mesitylimino-3-methyl-3,4-dihydro-2 (1H)-pyrimidone, on isolated guinea pig ventricular muscles and rabbit sinus node pacemaker cells were studied using micro-electrode techniques. In ventricular muscles driven at 0.5-1.0 Hz, FK664 above 3 mumol.litre-1 caused an increase in contractile force and a shortening of time to peak tension. This positive inotropic effect of FK664 was accompanied by a slight elevation of the early plateau phase of the action potential, while other action potential variables were unaffected. The change in contractile force induced by FK664 was abolished in a low Ca2+ medium (0.12 mmol.litre-1) or by treatment with ryanodine (2 mumol.litre-1), whereas it was relatively well preserved in the preparations pretreated with nefedipine (1 mumol.litre-1). The slow action potentials induced by isoprenaline (0.3 mumol.litre-1) in high K+ medium (30 mmol.litre-1) and the slow inward current measured by single sucrose gap voltage clamp at a holding potential of -40 mV were unaffected by FK664. In sinus node pacemaker cells, FK664 (1-10 mumol.litre-1) caused a dose dependent acceleration of phase 4 depolarisation and a shortening of spontaneous firing cycle length. This positive chronotropic effect of FK664 was markedly inhibited in a low Ca2+ medium (0.3 mmol.litre-1). These findings suggest that FK664 has positive inotropic and chronotropic effects on the heart, due to an enhancement of transsarcolemmal calcium influx through the low threshold, dihydropyridine insensitive Ca2+ channel population.

A field investigation of a modified intravaginal progesterone releasing device and oestradiol benzoate based ovulation synchronisation protocol designed for fixed-time artificial insemination of Brahman heifers.

Pregnancy rates (PR) to fixed-time AI (FTAI) in Brahman heifers were compared after treatment with a traditional oestradiol-based protocol (OPO-8) or a modified protocol (OPO-6) where the duration of intravaginal progesterone releasing device (IPRD) was reduced from 8 to 6 days, and the interval from IPRD removal to oestradiol benzoate (ODB) was increased from 24 to 36 h. Rising 2 yo heifers on Farm A: (n = 238 and n = 215; two consecutive days AI); B (n = 271); and C (n = 393) were allocated to OPO-8 or OPO-6. An IPRD was inserted and 1mg ODB i.m. on Day 0 for OPO-8 heifers and Day 2 for OPO-6 heifers. On Day 8, the IPRD was removed and 500 µg cloprostenol i.m. At 24h, for OPO-8 heifers, and 36 h, for OPO-6 heifers, post IPRD removal all heifers received 1mg ODB i.m. FTAI was conducted at 54 and 72 h post IPRD removal for OPO-8 and OPO-6 heifers. At Farm A, OPO-6 heifers, AI on the second day, the PR was 52.4% to FTAI (P = 0.024) compared to 36.8% for OPO-8 heifers. However, no differences were found between OPO-8 and OPO-6 protocols at Farm A (first day of AI) (39.9 vs. 35.7%), or Farms B (26.2 vs. 35.4%) and C (43.2% vs. 40.3%). Presence of a corpus luteum at IPRD insertion affected PR to FTAI (43.9% vs. 28.8%; P < 0.001). This study has shown that the modified ovulation synchronisation protocol OPO-6 may be a viable alternative to the OPO-8 protocol for FTAI in B. indicus heifers.

Phentolamine-induced rhythmic contractions in bladder detrusor muscle of guinea-pig.

Phentolamine caused a rhythmic contraction concentration-dependently without affecting resting tone in the detrusor muscle. Prazosin, yohimbine, propranolol, noradrenaline, clonidine or isoprenaline failed to cause the rhythmic contraction. These agents did not modify the response to phentolamine suggesting no involvement of alpha- or beta-adrenoceptors in the response to phentolamine. Chlorpheniramine, cimetidine, methysergide, SK&F 83566, atropine, bretylium, hemicholinium or tetrodotoxin failed to inhibit the response to phentolamine. These results suggest that the effect of phentolamine is not mediated through histaminergic, 5-hydroxytryptaminergic, dopaminergic or cholinergic systems, or through transmitter release from nerve endings. Prostaglandin F2 alpha (PGF2 alpha), arachidonic acid but not ATP caused rhythmic contractions which resembled the response to phentolamine. Potassium also caused a contraction with increasing resting tone. Following treatment with nifedipine, or incubation in a Ca2+-free medium, the responses to phentolamine, PGF2 alpha, arachidonic acid and potassium were markedly inhibited or abolished. Cyclo-oxygenase inhibitors such as indomethacin, aspirin and corticosterone inhibited or abolished the responses to phentolamine and arachidonic acid but did not inhibit the response to PGF2 alpha. The results suggest that the phentolamine-induced rhythmic contraction may, at least in part, result from the cyclo-oxygenase metabolite of arachidonic acid in guinea-pig detrusor muscles and a consequent increase in the transmembrane Ca2+-influx.

Mechanism of barium-induced contraction in the vascular smooth muscle of rabbit aorta.

In a solution containing 1.5 mM Ca2+, cumulative application of 0.3-10.0 mM Ba2+ induced a concentration-dependent contraction of the rabbit aorta. This contraction was reduced by the Ca2+ channel inhibitors, verapamil (10(-6) M), nifedipine (10(-7) M) and lanthanum (2.0 mM), and was potentiated by the Ca2+ channel facilitator, Bay K8644 (10(-7) M). In a Ca2+-free solution containing EGTA (1.0 mM), cumulative application of Ba2+ still induced a concentration-dependent contraction, the maximum contractile tension of which was comparable to that in the presence of 1.5 mM Ca2+. The Ba2+-induced contraction which was not dependent on the external Ca2+ was also inhibited by verapamil, nifedipine and lanthanum and was potentiated by Bay K8644. A high concentration (65.4 mM) of K+ potentiated this Ba2+-induced contraction whereas noradrenaline (10(-6) M) did not have such an effect. In order to deplete the releasable Ca2+ store in the cell, the muscle strip was treated with noradrenaline (10(-6) M) and/or caffeine (20.0 mM) in a Ca2+-free solution. In such a Ca2+-depleted muscle, Ba2+ still induced a contraction of a similar magnitude to that without such treatment. Further, the second application of Ba2+ in a Ca2+-free solution induced a similar contraction to that induced by the first application of Ba2+. These results suggest that Ba2+ depolarizes the cell membrane and opens the voltage-dependent Ca2+ channels resulting in a Ca2+ influx in the presence of Ca2+. In the absence of external Ca2+, Ba2+ may enter the cell through the voltage-dependent Ca2+ channels and induce contraction without mobilizing the Ca2+ store which is sensitive to noradrenaline and caffeine.

The inhibitory effects of iberiotoxin and 4-aminopyridine on the relaxation induced by beta 1- and beta 2-adrenoceptor activation in rat aortic rings.

1. In rat aortic rings contracted by phenylephrine, the relaxation induced by isoprenaline was partly inhibited by iberiotoxin, (ibTX), tetraethylammonium, 4-aminopyridine (4-AP) and 1,9-dideoxyforskolin, but not by glibenclamide. 2. In the presence of 4-AP, 1,9-dideoxyforskolin failed to inhibit further the relaxant response to isoprenaline. Cromakalim-induced relaxation was inhibited by glibenclamide. 3. In the absence of endothelium, ibTX and 4-AP still inhibited the relaxant response to isoprenaline. 4. The inhibitory effect of ibTX on the relaxant response to isoprenaline was eliminated by pretreatment with ICI-118,551, a beta 2-adrenoceptor antagonist, but not by atenolol, a beta 1-adrenoceptor antagonist. 5. The inhibitory effect of 4-AP on the relaxation induced by isoprenaline was abolished by atenolol, but not by ICI-118,551. 6. The inhibitory effect of ibTX on the isoprenaline-induced relaxation in the presence of atenolol was completely abolished by MDL 12,330A, an adenylate cyclase inhibitor. Further, the inhibitory effect of 4-AP on the isoprenaline-induced relaxation in the presence of ICI-118,551 was markedly reduced by MDL 12,330A. 7. The relaxation induced by dibutyryl cyclic AMP was partly inhibited by 4-AP but not by ibTX. However, in the presence of KT5720, an inhibitor of cyclic AMP-dependent protein kinase, ibTX failed to inhibit further the relaxation induced by isoprenaline. 8. These results suggest that, in rat aortic rings, KCa channels are involved in the relaxation induced by isoprenaline. In addition, KCa channels are mainly activated by beta 2-adrenoceptors through cyclic AMP-dependent pathways. Further, the inhibition of isoprenaline-relaxation by 4-AP may be related to the activation of beta 1-adrenoceptors and cyclic AMP formation.

Palytoxin-induced contraction and release of endogenous noradrenaline in rat tail artery.

1. The mechanism of the contractile effect of a potent marine toxin, palytoxin (PTX) on the rat isolated tail artery was examined. 2. PTX (10(-7) M) induced a contraction in the tail artery which was dependent on external Ca2+. This contraction was inhibited (by 75% or more) by 10(-6) M prazosin, 2.4 x 10(-5) M bretylium and 10(-4) M 6-hydroxydopamine (6-OHDA), and partially (by 40%) by 10(-5) M indomethacin. However, this contraction was not affected by 10(-6) M tetrodotoxin (TTX), 10(-6) M nifedipine or reserpine treatment. The PTX-induced contraction in reserpine-treated artery was partially inhibited by nifedipine and indomethacin but not by prazosin. 3. Transmural electrical stimulation induced a transient contraction which was dependent on external Ca2+. The contraction induced by electrical stimulation was inhibited by TTX, prazosin, bretylium, reserpine treatment and 6-OHDA but not by nifedipine or indomethacin. 4. PTX increased the release of noradrenaline from this artery. However PTX did not release noradrenaline from reserpine-treated arteries. PTX-induced noradrenaline release was only partially inhibited by TTX or by Ca2+-free solution. 5. These results suggest that PTX has pre- and postsynaptic effects in the rat tail artery. PTX may stimulate adrenergic nerves and release noradrenaline mainly by a TTX-insensitive and Ca2+-independent mechanism and partially by a TTX-sensitive and Ca2+-dependent mechanism. Further, PTX may also release prostaglandins and depolarize smooth muscle cell membrane to induce a contraction.

The mode of vasorelaxant action of 2-aminoisoquinoline, 1.3 (2H.4H)-dione, a novel 'intracellular calcium inhibitor'.

In rabbit aorta, pretreatment with 2-aminoisoquinoline, 1.3 (2H.4H)-dione (AQ, 10(-5) M and 10(-4) M) shifted the concentration-response relationship to noradrenaline (NA, 10(-9) M to 10(-4) M) in a parallel manner whereas the agent (10(-4) M) failed to affect the response to potassium and only slightly depressed Ca2+-induced contractions in a Ca2+-free medium in the presence of K+ (40 mM). Ca2+-entry blockers such as nifedipine and diltiazem (10(-6) M and 10(-5) M) had very weak or no apparent effects on the response to NA but markedly attenuated or abolished the K+- and Ca2+-induced contractions. Following incubation of tissues for 15 min in a Ca2+-free medium with low EGTA (0.01 mM) and methoxyverapamil (D600, 10(-5) M), NA (3 X 10(-7) M) caused a phasic (transient) contraction and the subsequent application of Ca2+ (2mM) resulted in a tonic contraction. This NA-induced, Ca2+-dependent, D600-insensitive contraction was inhibited by AQ (10(-5) M and 10(-4) M) in a concentration-dependent manner. This suggests that the inhibitory action of AQ may be related to Ca2+ entry through specific receptor activated pathways. Following incubation of tissues for 30 min in a Ca2+-free medium with high EGTA (2.0 mM), NA (10(-5) M) caused a contraction of rabbit aorta which is dependent upon release of intracellular Ca2+, but the response was 50% to 60% less than that in a normal medium. This contraction was inhibited by AQ (10(-5) M and 10(-4) M) and nitroglycerin (10(-5) M) but not by nifedipine or diltiazem. The inhibitory action of combined treatment with AQ and nitroglycerin (10-5 M) on the response to NA was not different from that of either agent alone. 5 These results suggest that AQ may have inhibitory actions on the release of intracellular Ca2+ and also on Ca2+-entry through D600-insensitive, receptor-activated Ca2+ pathways in rabbit aorta.

Possible mechanisms of inhibition with atropine against noradrenaline-induced contraction in the rabbit aorta.

1. In the rabbit isolated aorta, atropine (3 x 10(-6) M-10(-4) M) inhibited contractile response to noradrenaline without affecting contraction to KCl. 2. In the presence of contraction to noradrenaline, atropine (3 x 10(-7) M-10(-4) M) caused concentration-dependent relaxation. Pretreatment with theophylline (10(-3) M) potentiated the relaxant action of atropine. Relaxation to atropine was not affected by the specific guanosine 3':5'-cyclic monophosphate phosphodiesterase inhibitor, M & B 22,948 (10(-4) M), tetraethylammonium (10 mM), indomethacin (10(-5) M), propranolol (10(-7) M), nifedipine (10(-6) M) or removal of the endothelium. 3. Relaxation to either atropine or prazosin was not affected by preincubation with prazosin and atropine, respectively. 4. In Ca(2+)-free medium containing EGTA and nifedipine, atropine (10(-7) M-10(-4) M) inhibited the residual noradrenaline response more than the subsequent Ca(2+)-induced contraction. Pretreatment with either theophylline (10(-3) M), forskolin (3 x 10(-7) M) or a low concentration of prazosin (3 x 10(-9) M) also inhibited the residual contraction to noradrenaline and Ca2+. The effect of combined treatment of atropine and any of these agents was much greater than with each individual agent. 5. Atropine (10(-6) M-10(-4) M) also inhibited increases in the level of inositol monophosphates (IP) in response to noradrenaline. Theophylline (10(-3) M) and a low concentration of prazosin (3 x 10(-9) M) also inhibited IP formation. Combined with atropine, the effect was much greater than with each of these agents individually. 6. Atropine did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the aorta and also failed to displace specific [3H]-prazosin binding.7. These results suggest the possibility that smooth muscle relaxation to atropine may be due to the inhibition of phosphoinositide metabolism. The relaxation is not apparently due to an action of atropine on ax-adrenoceptors, or a change in the level of cyclic AMP.