The effects of firocoxib on cautery disbudding pain and stress responses in preweaned dairy calves.

Perioperative analgesic effects of oral firocoxib following cautery disbudding were investigated in preweaned calves. Twenty Holstein calves approximately 4 to 6wk old received a single oral dose of firocoxib, a nonsteroidal antiinflammatory, at 0.5mg/kg (n=10) or placebo (n=10) in a randomized controlled clinical trial. Responses, including ocular temperature determined by infrared thermography, pressure algometry measuring mechanical nociception threshold, and heart rate, were evaluated at 2, 4, 7, 8, and 24h after cornual nerve block and cautery disbudding. Blood samples were collected over 96h and analyzed for plasma cortisol and substance P concentrations by RIA. Additionally, ex vivo prostaglandin E2 concentrations were determined over a 72-h study period using an enzyme immunoassay. Data were analyzed using a linear mixed effects model with repeated measures. An inhibition of ex vivo prostaglandin E2 synthesis was observed from 12 to 48h following disbudding in calves treated with firocoxib. Cautery disbudding was associated with an increased nociception for the duration of sampling (24h). During the initial 24-h period following disbudding, no difference in response between treatment groups was noted. Following 24h, mean cortisol concentrations diverged between the 2 study groups with placebo-treated calves having increased cortisol concentrations at approximately 48h after disbudding. Furthermore, the overall integrated cortisol response as calculated as area under the effect curve tended to be reduced in firocoxib-treated calves. The prolonged effects of cautery dehorning require further investigation. Moreover, the effect of firocoxib on cortisol reduction observed in this study requires additional exploration.

Evaluating approaches to measuring ocular pain in bovine calves with corneal scarification and infectious bovine keratoconjunctivitis-associated corneal ulcerations.

Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease in cattle, associated with a 6.8 to 13.6 kg decrease in weaning weight. Antibiotic therapy is available but it is unclear if pain mitigation as an adjunct therapy would reduce the weight loss associated with IBK. Before assessing the impact of pain mitigation therapies, it is first necessary to validate approaches to qualifying ocular pain. The objective of this study was to evaluate approaches to qualifying ocular pain in bovine calves (Bos taurus) with IBK. Our a priori assumption was that scarification or corneal ulcerations consistent with IBK are painful compared to normal eyes. To quantify this difference in pain, we assessed 4 tools: pressure algometry-mechanical nociceptive threshold (PA-MNT), corneal touch thresholds (CTT) obtained with the use of a Cochet-Bonnet aesthesiometer, and assessment for the presence of blepharospasm and photophobia as metrics for pain. Using a 1-eye randomized controlled challenge trial, 31 calves with healthy eyes were randomly allocated to treatment groups, and then a left or right eye was randomly assigned for corneal scarification and inoculation with Moraxella bovoculi or Moraxella bovis. A repeated measures analysis of variance was used for PA-MNT, with significance set at P < 0.05. A log (base 10) transformation was used to stabilize the variance, and Tukey's t tests were used to test differences between assessment days for each landmark. Calves had statistically significantly lower PA-MNT scores (which indicates more pain) the day after scarification relative to baseline measurements (4 d before scarification). For example, at 1 landmark the median PA-MNT (kg/force) prescarification was 4.82 (95% confidence interval [CI]: 3.92-5.93) and 3.43 (95% CI: 2.79-4.22) postscarification. These data suggest PA-MNT may be a tool for quantifying ocular pain in calves. No differences (P < 0.1) in PA-MNT scores between scarified and not-scarified eyes were detected for any landmark on any day. This result suggests that the pain response occurs over the entire face, not just the affected eye. Corneal ulcerations consistent with IBK were not associated with statistically significant differences in PA-MNT or CTT at eye or calf levels. Not surprisingly, scarified eyes were more likely to exhibit blepharospasm and photophobia compared to healthy eyes. Due to blepharospasm, the use of the Cochet-Bonnet to evaluate corneal sensitivity by CTT was of limited value.

The economic value of organic dairy farms in Vermont and Minnesota.

This study quantifies the overall economic values of organic dairy farms in Vermont and Minnesota and estimates the economic impacts of organic dairy farm sales relative to an equivalent level of sales from conventional dairy farms in those states. This question is of interest because the development of the organic dairy sector has allowed some farms that likely would not have remained in the conventional dairy business to continue being economically viable as organic dairy farms. Thus, these sales provide an economic impact in regions when this milk is exported to nonproducing regions. Organic and conventional dairy farm financial data in Vermont and Minnesota were collected and assembled to develop dairy farm production functions by region and dairy type. These production functions were then used in state-level input-output models to calculate economic impacts. The opportunity costs of organic dairy farm production were also estimated by comparing the relative statewide economic impacts of organic and conventional dairy farms if both experience a hypothetical 5-million-dollar increase in sales. Between 2008 and 2010, Vermont's 180 organic dairy farms annually contributed $76.3 million in output (the value of an industry's production within the state), 808 jobs, $34.1 million in gross state product, and $26.3 million in labor income to Vermont's economy. Between 2009 and 2011, Minnesota's 114 organic dairy farms annually contributed $77.7 million in output, 552 jobs, $32.1 million in gross state product, and $21 million in labor income to Minnesota's economy. In Vermont, organic dairy farm sales revenue would result in greater state-wide impacts of 3% in output, 39% in labor income, 33% in gross state product, and 46% in employment relative to the impacts from an equivalent level of sales revenue to conventional dairy farms. In Minnesota, these economic impacts are 4, 9, 11, and 12% greater, respectively, for organic dairy farms relative to conventional dairy farms. This study concludes that organic dairy farms may contribute more to the local economy than average and similar-size conventional dairy farms in the Northeast and Upper Midwest and that organic dairy farm milk production supports economic development in rural communities.

Love thy neighbor--but does that include a six hundred eighty-four cow dairy operation? A survey of community perceptions.

The juxtaposition of nonfarming residences to operating dairy farms often precipitates conflict over appropriate land use. This was the situation facing the residents of the town of Charlotte, Vermont, in 2002 when a local dairy farmer proposed expanding from 225 to 684 cows with the construction of a new dairy facility and manure storage lagoon. The proposal raised considerable concern within the community and presented a unique opportunity for extension researchers to examine and analyze the attitudes and concerns of local residents toward the planned expansion, including their reasons for supporting or opposing the expansion, and to develop recommendations for farm operators considering future expansions. A survey instrument was developed and inserted in a local newspaper that was delivered to all households of Charlotte to identify important concerns of the community and explanatory factors differing between supporters and nonsupporters. Of those responding to the survey, 44.3% opposed the proposed dairy facility, 30.6% supported it, 17.9% needed more information to make a decision, and 7.2% had no opinion or were unaware of the proposal. There were no significant demographic (age, gender, educational attainment) differences between supporters and nonsupporters. Yet, the closer the proximity of the respondent's residence to the farm, the more likely he or she was to oppose it (beta = 1.018). The concerns of greatest importance were water quality (4.42/5), effect on property values (3.07/5), and animal welfare (3.58/5). Responses to the open-ended questions on the survey revealed strong views toward the farmer personally as well as concentrated animal feeding operations in general. The results indicate that farmers and extension need to take proactive steps to provide education and information relevant to the facts and issues surrounding new dairy facilities for 500 to 700 dairy cows.

A cost and returns evaluation of alternative dairy products to determine capital investment and operational feasibility of a small-scale dairy processing facility.

This study examines the economic feasibility of 50- and 500-cow dairy processing facilities for fluid milk, yogurt, and cheese. Net present value and internal rate of return calculations for projected costs and returns over a 10-yr period indicate that larger yogurt and cheese processing plants offer the most profitable prospects, whereas a smaller yogurt plant would break even. A smaller cheese plant would have insufficient returns to cover the cost of capital, and fluid milk processing at either scale is economically infeasible. Economic success in processing is greatly contingent upon individual business, financial management, and marketing skills.

Regulation of neuronal pituitary adenylate cyclase-activating polypeptide expression during culture of guinea-pig cardiac ganglia.

The trophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) increases in many different neuron types following injury; a response postulated to support cell survival and regeneration. In acutely isolated cardiac ganglia, approximately 1% of the cardiac neurons exhibited PACAP immunoreactivity whereas after 72 h in culture, approximately 25% of the neurons were PACAP immunoreactive. In contrast, there was no increase in vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) cells. Using a combination of immunocytochemical and molecular techniques, we have quantified PACAP expression, during explant culture of guinea-pig cardiac ganglia. Using real time polymerase chain reaction, PACAP transcript levels increased progressively up to 48 h in culture with no further increase after 72 h. PACAP transcript levels were reduced by neurturin at 48 h in culture but not after 24 or 72 h in culture. In addition, neurturin partially suppressed the percentage of PACAP-IR neurons after 72 h in culture, an effect mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways. The addition of different known regulatory molecules, including ciliary neurotrophic factor (CNTF), interleukin-1 beta (Il-1beta), tumor necrosis factor-alpha (TNFalpha), fibroblast growth factor basic (bFGF), transforming growth factor-beta (TGF-beta) and nerve growth factor (NGF) did not increase the percentage of PACAP-IR neurons after 24 h in culture; a result indicating that the generation and secretion of these factors did not stimulate PACAP expression. The presence of 20 nM PACAP or 10 muM forskolin increased the percentage of PACAP-IR cardiac neurons in 24 h cultures, but not in 72 h cultures. Neither treatment enhanced the number of VIP-IR neurons. The addition of the PACAP selective receptor (PAC(1)) receptor antagonist, M65 (100 nM) suppressed the 20 nM PACAP-induced increase in percentage of PACAP-IR cells in 24 h cultures indicating the effect of PACAP was mediated through the PAC(1) receptor. However, 100 nM M65 had no effect on the percentage of PACAP-IR cells in either 24 or 48 h cultures not treated with exogenous PACAP, suggesting that endogenous release of PACAP likely did not contribute to the enhanced peptide expression. We postulate that the enhanced PACAP expression, which occurs in response to injury is facilitated in the explant cultured cardiac ganglia by the loss of a target-derived inhibitory factor, very likely neurturin. In intact tissues the presence of neurturin would normally suppress PACAP expression. Lastly, our results indicate that many common trophic factors do not enhance PACAP expression in the cultured cardiac neurons. However, the stimulatory role of an, as yet, unidentified factor cannot be excluded.

Trophic factor modulation of cocaine- and amphetamine-regulated transcript peptide expression in explant cultured guinea-pig cardiac neurons.

The present study investigated the influence of trophic factors on the expression of cocaine- and amphetamine-regulated transcript peptide (CARTp) in guinea-pig cardiac ganglia maintained in explant culture. In acutely isolated cardiac ganglia preparations, <1% of the cholinergic cardiac neurons exhibited CARTp immunoreactivity. In contrast, this number increased to >25% of the cardiac neurons after 72 h in explant culture. This increase in the number of CARTp neurons in cultured cardiac ganglia explants was accompanied by an increase in CARTp transcript levels as assessed by real time polymerase chain reaction. Treatment of cardiac ganglia cultures with neurturin or glial-derived trophic factor (both at 10 ng/ml) for 72 h prevented the increase in neurons that exhibited CARTp immunoreactivity. In contrast, treatment with ciliary neurotrophic factor (50 ng/ml) for 72 h produced a small significant increase in the percentage of CARTp-immunoreactive cardiac neurons and treatment with nerve growth factor (100 ng/ml) had no effect. Neurturin treatment also decreased cardiac neuron CARTp levels after 72 h in explant culture. Cardiac neurons exhibited immunoreactivity to the neurturin receptor GFRalpha2 whereas non-neural cells preferentially exhibited immunoreactivity to the glial-derived neurotrophic factor receptor GFRalpha1 and neurturin transcripts were detected in cardiac tissue extracts. We hypothesize that a target-derived inhibitory factor, very likely neurturin, is a critical factor suppressing the expression of CARTp in guinea-pig cardiac neurons. These observations contrast with those reported in sympathetic neurons that suggest up-regulation of trophic factors after axotomy or during explant culture is a key factor contributing to the up-regulation of many neuropeptides.

Can we identify key characteristics associated with grazing-management dairy systems from survey data?

Discriminant analysis was used to identify farms using confinement and grazing-production systems from mail survey data of 2074 dairy farmers in Pennsylvania, Vermont, Virginia, and North Carolina. Survey respondents included 45.1% of the farms using confinement management; 13.5% of farms practicing intensive grazing, defined as moving cows to new pasture at least every 3 d; and 41.4% of farms using nonintensive grazing. Farmers using confinement management had significantly more cows, higher milk production, more crop acreage, higher debt, used automatic takeoff milking units (ATO), fed total mixed rations (TMR), and were more satisfied. In general, dairy farmers who grazed their milking cows had smaller herds, fewer acres, but had more acres per cow and made less use of technology. However, farmers practicing intensive grazing were significantly younger, more educated, less experienced, more likely to use computers, and farmed less acreage than other graziers or farmers on confinement farms. The discriminant function correctly classified 70% of the total sample when divided into confinement and overall grazing categories. However, the discriminant function correctly classified only 36% of intensive-grazing farms in comparison to confinement farms. Significant variables identified using ordinary least squares as being related to confinement management were milk per cow, acres of corn, use of ATO and TMR, debt greater than 40%, and residence in North Carolina. Significant variables associated with grazing management were acres of pasture, future use of pasture, education, and residence in Vermont. The analysis indicated that the discriminant function could correctly classify confinement and nonintensive-grazing management but was unable to reliably differentiate between confinement and intensive-grazing farms.

Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia.

Postganglionic parasympathetic neurons in guinea-pig cardiac ganglia exhibit choline acetyltransferase (ChAT)-immunoreactivity, and a large fraction (60%) of the ChAT-positive cardiac neurons co-express somatostatin-immunoreactivity. This co-expression remained when the cardiac ganglia explants were maintained in culture for 72 h (40% somatostatin-immunoreactive). The guinea-pig cardiac ganglia neurons express the high affinity pituitary adenylate cyclase activating polypeptide (PACAP)-selective PAC1 receptor, and treatment of the ganglia explants with 20 nM PACAP27 for 72 h to evaluate PACAP regulation of somatostatin expression revealed a dramatic 85% decrease in the number of somatostatin-IR neurons (6% somatostatin-IR neurons) compared with untreated control explant preparations. The decrease in percentage of somatostatin-IR neurons by PACAP27 was time- and concentration-dependent, and selective for PACAP27; PACAP38 and vasoactive intestinal polypeptide were less effective. PACAP6-38, a PACAP antagonist, eliminated the PACAP27-induced change in somatostatin positive neurons. The PACAP-mediated decrease in somatostatin-IR neurons was eliminated in calcium-deficient solutions and by the addition of nifedipine, indicating a requirement for calcium influx through L-type calcium channels. The addition of either the calmodulin inhibitor N-(4-aminobutyl)-1-naphthalenesulfonamide or the MEK inhibitor PD98059, also eliminated the PACAP27-induced decrease in somatostatin-IR cells. The PACAP27-mediated effect on somatostatin expression was not affected by inhibitors of protein kinase A or phospholipase C, but was reduced by the adenylyl cyclase inhibitor SQ22356, suggesting cAMP involvement. Semiquantitative and quantitative reverse transcription PCR prosomatostatin transcript measurements showed that cardiac ganglia prosomatostatin mRNA levels were not diminished by chronic PACAP27 exposure despite the dramatic decrement in somatostatin-expressing neurons. Neuronal peptide-IR content represents a balance between production and secretion. These results suggested that one of the primary effects of PACAP exposure may be enhanced levels of neuropeptide release that exceeded production levels, resulting in somatostatin depletion and a decrement in the number of identifiable somatostatin-expressing cardiac neurons.

The modulation of action potential generation by calcium-induced calcium release is enhanced by mitochondrial inhibitors in mudpuppy parasympathetic neurons.

Previously, we demonstrated that outward currents activated by calcium-induced calcium release (CICR) opposed depolarization-induced action potential (AP) generation in dissociated mudpuppy parasympathetic neurons [J Neurophysiol 88 (2002) 1119]. In the present study, we tested whether AP generation by depolarizing current ramps could be altered by dissipating the mitochondrial membrane potential and thus interrupting mitochondrial Ca2+ buffering. Exposure to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 microM) alone or in combination with the mitochondrial ATP synthase inhibitor oligomycin (8 microg/ml), increased the latency to AP generation. Exposure to the electron transport chain inhibitor rotenone (10 microM) alone or in combination with oligomycin (8 microg/ml) similarly increased the latency to AP generation. CCCP and oligomycin or rotenone and oligomycin treatment caused rhodamine 123 loss from mitochondria within a few minutes, confirming that the mitochondrial membrane potential was dissipated during drug exposure. Oligomycin alone had no effect on the latency to AP generation and did not cause loss of rhodamine 123 from mitochondria. The increase in latency induced by CCCP and oligomycin was similar when recordings were made with either the perforated patch or standard whole cell patch recording configuration. Exposure to the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (1 microM), decreased the latency to AP generation. In cells pretreated with thapsigargin to eliminate CICR, CCCP and oligomycin had no effect on AP latency. Pretreatment with iberiotoxin (IBX; 100 nM), an inhibitor of large conductance, calcium- and voltage-activated potassium channels, reduced the extent of the CCCP- and oligomycin-induced increase in latency to AP generation. These results indicate that treatment with CCCP or rotenone to dissipate the mitochondrial membrane potential, a condition which should minimize sequestration of Ca2+ by mitochondria, facilitated the Ca(2+)-induced Ca2+ release activation of IBX-sensitive and IBX-insensitive conductances that regulate AP generation.

In vivo three-dimensional imaging of plants with optical coherence microscopy.

Achieving the ability to non-destructively, non-invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics-based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three-dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non-invasive, non-destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.

Innervation of guinea-pig stellate ganglia by nitric oxide synthase, cocaine- and amphetamine-regulated transcript protein- and pituitary adenylate cyclase activating polypeptide-immunoreactive fibers.

The present study analyzed using immunohistochemical labeling the distribution and co-localization of nitric oxide synthase (NOS), cocaine- and amphetamine-regulated transcript peptide (CARTp) and pituitary adenylate cyclase activating polypeptide (PACAP) with choline acetyltransferase (ChAT)-immunoreactive fibers in the guinea-pig stellate ganglia. ChAT-immunoreactive fibers make pericellular baskets around virtually all stellate ganglia neurons. Pericellular baskets of NOS, CARTp and PACAP fibers were also present around numerous stellate ganglia neurons. Although all the NOS and PACAP fibers also exhibited ChAT immunoreactivity, only some of the CARTp fibers were ChAT-immunoreactive. No evidence of co-localization of NOS, PACAP and CARTp was obtained.These results indicate that NOS, PACAP and CARTp are present in distinct preganglionic axons innervating the guinea-pig stellate ganglia.

Distribution of cocaine- and amphetamine-regulated transcript peptide in the guinea pig intrinsic cardiac nervous system and colocalization with neuropeptides or transmitter synthetic enzymes.

This study was conducted to establish the presence of cocaine- and amphetamine-regulated transcript peptide (CARTp) immunoreactivity in neurons and fibers within guinea pig atrial whole-mount preparations containing the intrinsic cardiac ganglia. Many cardiac ganglia, but not all, in a given whole-mount preparation, were innervated by CARTp-immunoreactive (IR) fibers. Following explant culture of whole mounts for 72 hours, the CARTp-IR fiber networks were absent, but the number of CARTp-IR neurons was increased markedly. These observations suggested that the majority of the CARTp-IR fibers in the intracardiac ganglia were derived from sources extrinsic to the heart. In control whole-mount preparations, very few CARTp-positive neurons were present. The few intrinsic CARTp-IR neurons also exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they make up a small subpopulation of cholinergic postganglionic neurons. Some CARTp-IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of CARTp-IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs. The CARTp-IR fibers were not immunoreactive for ChAT, tyrosine hydroxylase, calcitonin gene-related peptide, or substance P. However, virtually all CARTp-IR fibers exhibited immunoreactivity to neuronal NOS (a marker for nitric oxide-producing neurons). CARTp-IR cells and NOS-IR cells were present in the nodose ganglia. In addition, CARTp-IR neurons in the nodose also were stained positively for NADPH-diaphorase. Thus, we propose that most CARTp-IR fibers within the guinea pig intrinsic cardiac ganglia are vagal afferent fibers that also contain NOS.

NMR spectroscopic investigations of mixed aggregates underlying highly enantioselective 1,2-additions of lithium cyclopropylacetylide to quinazolinones.

The solution structures of mixed aggregates derived from lithium alkoxides and lithium acetylides were investigated as part of a program to develop practical syntheses of quinazolinone-based nonnucleoside reverse transcriptase inhibitors. Low-temperature (6)Li, (13)C, and (15)N NMR spectroscopies reveal that mixtures of lithium cyclopropylacetylide (RCCLi), a (+)-carene-derived amino alkoxide (ROLi), and lithium hexamethyldisilazide (LiHMDS) in THF/pentane afford a (RCCLi)(3)(ROLi) mixed tetramer, a C(2)-symmetric and asymmetric (RCCLi)(2)(ROLi)(2) mixed tetramer, and a C(3)-symmetric (RCCLi)(ROLi)(3) mixed tetramer. Analogous mixtures of RCCLi/ROLi in Et(2)O and Me(2)NEt also provide 3:1, 2:2, and 1:3 mixed tetramers. The stereochemistry of aggregation is highly sensitive to the medium. The C(2)-symmetric (RCCLi)(2)(ROLi)(2) mixed tetramer is formed in Et(2)O, whereas the asymmetric isomer is formed in Me(2)NEt. LiHMDS in THF is shown to be an efficient proton scavenger without forming LiHMDS-RCCLi or LiHMDS-ROLi mixed aggregates. LiHMDS-RCCLi mixtures form mixed aggregates in Me(2)NEt.

Synthesis and evaluation of efavirenz (Sustiva) analogues as HIV-1 reverse transcriptase inhibitors: replacement of the cyclopropylacetylene side chain.

Two series of efavirenz analogues have been developed: one in which the cyclopropane ring has been replaced by small heterocycles and another in which the entire acetylenic side chain has been replaced by alkyloxy groups. Several members of both series show equivalent potency to efavirenz against both wild-type virus and the key K103N mutant.

Presynaptic function is altered in snake K+-depolarized motor nerve terminals containing compromised mitochondria.

Presynaptic function was investigated at K+-stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 M), oligomycin (8 g x ml(-1)) or CCCP and oligomycin together. Miniature endplate currents (MEPCs) were recorded at -150 mV with two-electrode voltage clamp. With all three drug treatments, during stimulation by elevated K+ (35 mM), MEPC frequencies initially increased to values > 350 s(-1), but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone. Staining with FM1-43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP- or oligomycin-treated nerve terminals after 120 or 180 min of K+ stimulation, respectively. The addition of glucose to stimulate production of ATP by glycolysis during sustained K+ stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1-43 in preparations exposed to either CCCP or oligomycin. We propose that the decline in K+-stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca2+]i). For oligomycin-treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy-dependent Ca2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca2+]i could occur more rapidly.

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons.

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.

An efficient chiral moderator prepared from inexpensive (+)-3-carene: synthesis of the HIV-1 non-nucleoside reverse transcriptase inhibitor DPC 963.

The beta-amino alcohol 4 beta-morpholinocaran-3 alpha-ol is prepared by addition of morpholine to alpha-3,4-epoxycarane utilizing anhydrous magnesium bromide as Lewis acid promoter. The enantiopure amino alcohol is uniquely effective as a chiral moderator for the addition of lithium cyclopropylacetylide to an unprotected N-acylketimine. This reaction provides an efficient route to the second generation NNRTI drug candidate DPC 963.

Mechanisms mediating pituitary adenylate cyclase-activating polypeptide depolarization of rat sympathetic neurons.

The direct effects of pituitary adenylate cyclase-activating polypeptides (PACAP) on sympathetic neurons were investigated using rat superior cervical ganglion neurons. Electrophysiological and pharmacological analyses were used to evaluate PACAP modulation of sympathetic neuron membrane potentials and to investigate potential ionic and intracellular signaling mechanisms mediating the responses. More than 90% of the sympathetic neurons were depolarized by the PACAP peptides even when stimulated release was blocked, indicating that the PACAP peptides elicited primary responses in the postganglionic neurons. The response profile was consistent for activation of PACAP-selective PAC(1) receptors: nanomolar concentrations of PACAP27 and PACAP38 were required to stimulate depolarization, whereas vasoactive intestinal peptide failed to evoke any response. Furthermore, depolarizations elicited by PACAP27 were reduced by the PAC(1) receptor antagonist PACAP(6-38). Both sodium influx and inhibition of a potassium current contributed to the peptide-induced depolarizations. Activation of neither pertussis toxin- nor cholera toxin-sensitive G-proteins was required for generation of the depolarizations. cAMP and diacylglycerol production and activation of protein kinase A or protein kinase C also were not requisite for the responses. By contrast, phospholipase C (PLC)-dependent inositol 1,4,5-triphosphate (IP(3)) synthesis was crucial to the PACAP-mediated depolarizations. Although calcium release from IP(3)-sensitive stores was not required for the PACAP-induced responses, inhibition of IP(3) receptors reduced the depolarizations. Thus, among the many signal transduction pathways coupled to the PAC(1) receptor, the PACAP-induced depolarization of sympathetic neurons appears to require activation of PLC and subsequent generation of IP(3).

Origin of neuronal nitric oxide synthase (NOS)-immunoreactive fibers in guinea pig parasympathetic cardiac ganglia.

This study was conducted to determine the origin(s) of neuronal nitric oxide synthase-immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations containing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Comparison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1-5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia.