Disease epidemic and a marine heat wave are associated with the continental-scale collapse of a pivotal predator (Pycnopodia helianthoides).

Multihost infectious disease outbreaks have endangered wildlife, causing extinction of frogs and endemic birds, and widespread declines of bats, corals, and abalone. Since 2013, a sea star wasting disease has affected >20 sea star species from Mexico to Alaska. The common, predatory sunflower star (Pycnopodia helianthoides), shown to be highly susceptible to sea star wasting disease, has been extirpated across most of its range. Diver surveys conducted in shallow nearshore waters (n = 10,956; 2006-2017) from California to Alaska and deep offshore (55 to 1280 m) trawl surveys from California to Washington (n = 8968; 2004-2016) reveal 80 to 100% declines across a ~3000-km range. Furthermore, timing of peak declines in nearshore waters coincided with anomalously warm sea surface temperatures. The rapid, widespread decline of this pivotal subtidal predator threatens its persistence and may have large ecosystem-level consequences.

P2Y2 receptor activation decreases blood pressure via intermediate conductance potassium channels and connexin 37.

AIMS: Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y2 receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y2 receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response. METHODS: We compared the effects of a P2Y2/4 receptor agonist in wild-type (WT) mice and in mice lacking KCa3.1, KCa1.1, Cx37 or Cx40 under anaesthesia, while monitoring intra-arterial blood pressure and heart rate. RESULTS: Acute activation of P2Y2/4 receptors (0.01-3 mg kg(-1) body weight i.v.) caused a biphasic blood pressure response characterized by a dose-dependent and rapid decrease in blood pressure in WT (maximal response % of baseline at 3 mg kg(-1) : -38 ± 1%) followed by a consecutive increase in blood pressure (+44 ± 11%). The maximal responses in KCa3.1(-/-) and Cx37(-/-) were impaired (-13 ± 5, +17 ± 7 and -27 ± 1, +13 ± 3% respectively), whereas the maximal blood pressure decrease in response to acetylcholine at 3 µg kg(-1) was not significantly different (WT: -53 ± 3%; KCa3.1(-/-) : -52 ± 3; Cx37(-/-) : -53 ± 3%). KCa1.1(-/-) and Cx40(-/-) showed an identical biphasic response to P2Y2/4 receptor activation compared to WT. CONCLUSIONS: The data suggest that the P2Y2/4 receptor activation elicits blood pressure responses via distinct mechanisms involving KCa3.1 and Cx37.

Parental identity influences progeny responses to incubation thermal stress in sockeye salmon Onchorhynchus nerka.

The influence of individual parentage on progeny responses to early developmental temperature stress was examined in a cross-fertilization experiment using sockeye salmon Oncorhynchus nerka. Differences in survival, hatch timing and size were examined among five paternally linked and five maternally linked offspring families (Weaver Creek population, British Columbia, Canada) incubated at 12, 14 and 16° C from just after fertilization to hatch. Mean embryonic survival was significantly lower at 14 and 16° C; however, offspring families had substantially different survival responses across the thermal gradient (crossing reaction norms). Within temperature treatments, substantial variation in embryonic survival, alevin mass, time-to-hatch and hatch duration were attributable to family identity; however, most traits were governed by significant temperature-family interactions. For embryonic survival, large differences between families at 16° C were due to both female and male spawner influence, whereas inter-family differences were obscured at 14° C (high intra-family variation), and minimal at 12° C (only maternal influence detected). Despite post-hatch rearing under a common cool thermal regime, persistent effects of both temperature and parentage were detected in alevin and 3 week-old fry. Collectively, these findings highlight the crucial role that parental influences on offspring may have in shaping future selection within salmonid populations exposed to elevated thermal regimes. An increased understanding of parental and temperature influences and their persistence in early development will be essential to developing a more comprehensive view of population spawning success and determining the adaptive capacity of O. nerka populations in the face of environmental change.

Temporal changes in blood variables during final maturation and senescence in male sockeye salmon Oncorhynchus nerka: reduced osmoregulatory ability can predict mortality.

This study is the first to characterize temporal changes in blood chemistry of individuals from one population of male sockeye salmon Oncorhynchus nerka during the final 6 weeks of sexual maturation and senescence in the freshwater stage of their spawning migration. Fish that died before the start of their historic mean spawning period (c. 5 November) were characterized by a 20-40% decrease in plasma osmolality, chloride and sodium, probably representing a complete loss of osmoregulatory ability. As fish became moribund, they were further characterized by elevated levels of plasma cortisol, lactate and potassium. Regressions between time to death and plasma chloride (8 October: P < 0·001; 15 October: P < 0·001) indicate that plasma chloride was a strong predictor of longevity in O. nerka. That major plasma ion levels started to decline 2-10 days (mean of 6 days) before fish became moribund, and before other stress, metabolic or reproductive hormone variables started to change, suggests that a dysfunctional osmoregulatory system may initiate rapid senescence and influence other physiological changes (i.e. elevated stress and collapsed reproductive hormones) which occur as O. nerka die on spawning grounds.

Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons.

Recent evidence indicating formation of functional homomeric/heterotypic gap junction channels by connexin40 (Cx40) and connexin43 (Cx43) raises the question of whether data previously interpreted as support for heteromeric channel formation by these connexins might not instead reflect the activity of homomeric/heterotypic channels. To address this question and to further characterize the behavior of these channels, we used dual whole cell voltage-clamp techniques to examine the junctions formed between cells that express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired with cells that naturally express both connexins (A7r5 cells). Rin40/Rin43 cell pairs formed functional gap junctions that displayed a strongly asymmetric voltage-dependent gating response. Single-channel event amplitudes ranged between 34 and 150 pS, with 90- to 130-pS events predominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairs demonstrating strong asymmetry. These cell pairs exhibited a variety of single-channel events that were not consistent with homomeric/homotypic Cx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels. These data indicate that Cx40 and Cx43 form homomeric/heterotypic as well as heteromeric/heterotypic channels that display unique gating and conductance properties.

Alteration of Cx43:Cx40 expression ratio in A7r5 cells.

Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.

Functional characteristics of heteromeric Cx40-Cx43 gap junction channel formation.

Cx40:Cx43 expression ratio in A7r5 cells is augmented in growth stimulated vs. growth arrested conditions. To determine the impact of changing Cx40:Cx43 expression ratio on gap junction function, we have developed A7r5 cell lines that display Cx40:Cx43 ratios of 1:1 (66B5n) and 10:1 (A7r540C3). When Rin43 cells were paired with these coexpressing cells, there was an increasing asymmetry of voltage dependent gating as the Cx40:Cx43 ratio increased in the coexpressing cell. This asymmetry was opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In addition, when Rin43 cells were paired with coexpressing cells there was a shift toward smaller single channel event amplitudes with increasing Cx40:Cx43 ratio in the coexpressing cell. Again, this is opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In dye coupling experiments, 6B5N, A7r5, and A7r540C3 cells displayed charge and size selectivity that increased with increasing Cx40:Cx43 expression ratio. These data indicate that although the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents that comprise the channel, the dye permeability data fit what would be predicted by an increase in Cx40:Cx43 ratio.

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

Mechanism and selectivity of the effects of halothane on gap junction channel function.

Volatile anesthetics alter tissue excitability by decreasing the extent of gap junction-mediated cell-cell coupling and by altering the activity of the channels that underlie the action potential. In the present study, we demonstrate, using dual whole-cell voltage-clamp techniques, that coexpression of connexin (Cx) 40 and Cx43 rendered cells more sensitive to uncoupling by halothane than cells that express only Cx40 or only Cx43. The halothane-induced reduction in junctional conductance was caused by decreased channel mean open time and increased channel mean closed time. The magnitude of the effect of halothane on channel open time was least for Cx40-like channels and greatest for heteromeric channels. Thus, the data indicate that halothane gates gap junction channels to the closed state in a dose-dependent and connexin-specific manner. One consequence of the selectivity of halothane is that the profile of single-channel events observed in the presence of halothane may not be quantitatively representative of the population of channels contributing to macroscopic conductance in cells that express more than one connexin. In addition, in tissues that express multiple connexins, such as heart and blood vessels, the capacity of the gap junctions to transmit electrical and chemical signals in the presence of halothane could vary according to the pattern of connexin expression.

Lesions of the anterior forebrain song control pathway in female canaries affect song perception in an operant task.

We tested whether the avian anterior forebrain pathway functions in song perception in female canaries, and whether it is specialized for conspecific song perception or functions more generally in auditory perception. Using operant conditioning methods, we trained female canaries to discriminate among synthetic sound stimuli, canary songs, and song sparrow songs. We also trained each bird to discriminate among visual stimuli to test for general effects of lesions on performance. When canaries had learned the discrimination tasks, bilateral electrolytic lesions of the lateral portion of the magnocellular nucleus of the anterior neostriatum (lMAN) were made. The lesioned birds were then tested on the previously learned discrimination tasks. Lesions that destroyed most or all of lMAN decreased the ability of female canaries to discriminate between previously learned pairs of acoustic stimuli of all types, while visual discrimination was unaffected. These results suggest that the female canary anterior forebrain pathway contributes to the perception of acoustic stimuli, with this contribution including heterospecific song and other acoustic stimuli as well as canary song.

Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells.

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

Gap junction permeability is diminished in proliferating vascular smooth muscle cells.

In atherosclerosis and hypertension, vascular smooth muscle cells (SMCs) are stimulated to proliferate and exhibit enhanced gap junction protein expression. Our goal was to determine whether gap junction function differs in proliferating vs. growth-arrested SMCs. A7r5 cells (embryonic rat aortic SMCs) did not proliferate in media with reduced serum ( approximately 90% of cells in G0/G1 phase after 48-96 h in 1% fetal bovine serum). Dye coupling was less but electrical coupling was comparable in proliferating vs. growth-arrested A7r5 cells, suggesting differences in junctional permselectivity. In growth-arrested cells, junctional conductances measured with potassium glutamate, tetraethylammonium chloride, and KCl were well predicted by the conductivities of these solutions. In contrast, junctional conductances measured with potassium glutamate and tetraethylammonium chloride in proliferating cells were significantly greater than predicted by the conductivities of these solutions. These results suggest that junctions between growth-arrested cells are permeated equally well and simultaneously by anions and cations, whereas junctions between proliferating cells are poorly permeated by large molecules of either charge and equally well but not simultaneously by small anions and cations. The data indicate that A7r5 cells regulate chemical coupling independent of electrical coupling, a capacity that could facilitate growth control while protecting vasomotor responsiveness of vessels.

Auditory perception following hair cell regeneration in European starling (Sturnus vulgaris): frequency and temporal resolution.

Behavioral detection thresholds, auditory filter widths, and temporal modulation transfer functions were obtained from four starlings before, during and after 11 days of subcutaneous injections of kanamycin, an aminoglycoside antibiotic. Birds were operantly conditioned to respond to pure tones and amplitude modulated noises ranging in frequency from 0.25 to 7 kHz using adaptive staircase procedures and were tested daily for 92 days after the first injection of aminoglycoside. All birds had threshold shifts of at least -60 dB at frequencies above 4 kHz. Lower frequencies were affected in some birds, although none of the birds had hearing loss below 3 kHz. All four birds had wider auditory filters at 5 kHz immediately after the aminoglycoside series. Any changes in frequency resolution at frequencies below 5 kHz were slight, transitory, and rarely observed. Two of the four birds had permanently wider auditory filters at 5 kHz. Temporal modulation transfer functions were briefly affected in two birds during the time of greatest threshold shift. Recovery of detection thresholds began soon after the injections ceased and continued for approximately 60 days. Recovery in frequency resolution lagged behind auditory threshold by about 10 days. Normal temporal resolution was observed in the context of impaired intensity and frequency resolution. Changes in auditory threshold and frequency resolution were closely associated for all birds at 5 kHz, but were correlated with statistical significance in only two birds. Scanning electron microscopy was performed on all four birds after 90 days of recovery and confirmed that the extent of initial damage was consistent with the pattern of observed hearing loss.

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase.

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.

Immortalized connexin43 knockout cell lines display a subset of biological properties associated with the transformed phenotype.

Immortalized cells from embryonic connexin43 knockout mice (Cx43-/-) and homozygous littermates (Cx43+/+) were cloned and characterized to determine whether the absence of Cx43 function would induce observable phenotypic changes. Cells of the Cx43+/+ clones expressed Cx43 and engaged in gap junctional communication with 10-12 neighboring cells. The Cx43-/- cells were devoid of Cx43 and communicated to less than 1 cell. Electrophysiological analysis indicated that the Cx43-/- cells communicated through Cx45 channels from 8-80-fold less than did the Cx43+/+ subclones, which seemed to communicate through Cx43 and Cx45 channels. The Cx43-/- clones grew at faster rates and to higher saturation densities, had a more spindly morphology, were more refractile, and adhered less well to the substratum than did the Cx43+/+ clones. Reintroducing the Cx43 gene into the Cx43-/- clones resulted in three subclones that communicated to 3-4 cells. Partial restoration of gap junctional communication in the three subclones was accompanied by reduced growth rates and saturation densities (2-fold compared to that of parental Cx43-/- clones) but no reversions in morphology or cell-substratum adhesion. The increased growth rates and saturation densities, altered morphology, and decreased cell adhesion displayed by the Cx43-/- clones reflect a subset of the properties of transformed cells. These studies advance the hypothesis that loss of Cx43 function during development may cause cells to acquire a preneoplastic condition.

Gap junction function in vascular smooth muscle: influence of serotonin.

In this study we examined the effects of serotonin (5-hydroxytryptamine, 5-HT) on the function of gap junctions between smooth muscle cells isolated from human and pig coronary and rat mesentery arteries and between A7r5 cells (cell line derived from embryonic rat aorta). Mesentery and pig coronary cells expressed connexin (Cx) 43, and human coronary cells expressed Cx40. Mesentery and pig coronary cells each exhibited a single gap junction channel population with unitary conductances of 75 and 59 pS, respectively. Human coronary cells exhibited two channel populations with unitary conductances of 51 and 107 pS. The A7r5 cells express Cx40 and Cx43 and exhibit three channel populations with unitary conductances of 70, 108, and 141 pS. Under control conditions, junctional conductance between the four cell types ranged from 11 to 20 nS. During maximal stimulation with 5-HT (1-10 microM), junctional conductance increased (29-75%) in all four cell types. The unitary conductance profiles in the rat mesentery and pig coronary cells were unaffected by 5-HT, suggesting that the observed increase in macroscopic conductance reflects an increase in open probability. Unitary conductances were also unaffected in the human coronary and A7r5 cells. However, there was a reduced frequency of the 105-pS channel in the human coronary cells and of the 70- and 141-pS channels in the A7r5 cells. These changes in the relative frequency histograms suggest that the open probabilities of the various channel types are differentially affected by the 5-HT treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Regenerated hair cells in the European starling: are they more resistant to kanamycin ototoxicity than original hair cells?

Previous work from our laboratory [Marean et al. (1993) Hear. Res. 71, 125-136] has shown that a 10 day dose of 200 mg/kg/day kanamycin produced damage to the basal 34% of the starling basilar papilla. We also observed that repeating the dosing schedule following a 4 month survival period resulted in significantly less damage to the regenerated auditory epithelium. The present study investigated whether or not this apparent resistance was the result of a tendency for regenerated hair cells to be less susceptible to kanamycin ototoxicity, or if other, systemic factors may be involved. Eight European starlings were given subcutaneous injections of 200 mg/kg/day kanamycin for 10 days. Serum levels of kanamycin were measured at the time of sacrifice for all birds, and the basilar papillae of all birds were examined by scanning electron microscopy (SEM). Two of these birds (Group 1) were sacrificed immediately following the dosing period. Two of the birds were allowed to survive for 60 days (Group 2). Two of the birds were redosed with 200 mg/kg/day for 10 days after 60 days survival (Group 3). Finally, two birds were redosed with 250 mg/kg/day until serum levels of kanamycin were the same as Group 1 when sacrificed (> 9 micrograms/ml). The SEM results showed that the regenerated auditory epithelium of the birds dosed a second time sustained less damage compared to previously untreated ears, even though the dosing regimen was the same (Group 3 versus Group 1). The regenerated auditory epithelium of birds dosed a second time sustained the same damage as previously untreated animals when the dosage was increased to attain similar serum levels (Group 4 versus Group 1). These results suggest metabolic changes occur in the starling in response to the initial dose of kanamycin which do not necessarily involve changes in hair cell resistance to ototoxicity.

Selective block of gap junction channel expression with connexin-specific antisense oligodeoxynucleotides.

Gap junctions in vascular smooth muscle provide a cell-to-cell conduction pathway that may contribute to regulation and coordination of changes in vascular tone. A7r5 cells, a cell line derived from embryonic rat aorta, express connexin 43 (Cx43) and connexin 40 (Cx40). Gap junction channels with three distinct unitary conductances (70, 108, and 141 pS) were observed in these cells. Events of each channel type were equally common, with an approximate frequency of 30-35%; however, the frequency at which each channel type was observed in individual cell pairs ranged between 10 and 65%. Treatment of the cells for 24-72 h with an antisense oligodeoxynucleotide (ODN) to Cx43 reduced the relative frequency of the 108- and 141-pS channel events, whereas comparable treatment with antisense Cx40 ODN reduced the frequency at which 70-pS channel events were observed. The simplest explanation of these findings is that Cx43 forms the 108- and 141-pS channels, whereas Cx40 forms the 70-pS channels in A7r5 cells.

Hair cell regeneration in the European starling (Sturnus vulgaris): recovery of pure-tone detection thresholds.

Behavioral detection thresholds were obtained from four starlings before, during, and after 11 days of subcutaneous injections of kanamycin. Birds were operantly conditioned to respond to pure-tones ranging in frequency from 0.25 kHz to 7 kHz using the method of constant stimuli and were tested daily for 141 days after the first injection of aminoglycoside. All four birds sustained hearing losses greater than 60 dB at frequencies from 4 kHz to 7 kHz by the end of the 11 day injection schedule. Two birds had a slight shift in threshold at 3 kHz. No change in threshold occurred for any of the birds at lower frequencies. Recovery of detection thresholds began soon after the injections ceased and continued for approximately 50 days. In all four birds there was some degree of permanent hearing loss: 5 dB to 15 dB at frequencies between 4 kHz and 6 kHz, and approximately 25 dB at 7 kHz. Scanning electron microscopy (SEM) was performed at 0 and 5 days post-injection in a separate group of starlings given the same injection schedule. Hair cell loss and damage was observed across the basal 34% to 36% of the basilar papilla. SEM in two behaviorally tested birds sacrificed 142 days after the first injection showed that there was regeneration of hair cells to populate the previously damaged region, but that disorientation of stereocilia bundles in the basal third of the basilar papilla was common. The other two behaviorally tested birds were treated with kanamycin again for 16 days beginning at 142 days after the first injection. Thresholds shifted again, but less than during the first dosing period. SEM of these birds' basilar papillae showed less hair cell loss than observed in the birds given only a single, 11 day dosing of kanamycin. This result suggests that birds may be less susceptible to the ototoxic effects of kanamycin in repeated treatments. In all four birds, the degree and position of damage observed with SEM corresponded with the extent and frequency of hearing loss.

Oleic acid differentially affects gap junction-mediated communication in heart and vascular smooth muscle cells.

The effects of oleic acid (OA) on gap junction-mediated intercellular communication between A7r5 cells and neonatal rat cardiac myocytes were determined. In A7r5 cells the extent of dye coupling was influenced in a biphasic manner by increasing concentrations of OA. Low concentrations of OA (0.1-1 microM) reduced the incidence of dye coupling from 90% (in control cells) to approximately 50%. Further increases in OA concentration, up to 100 microM, had no further effect on extent of dye coupling. In contrast, dye coupling between cardiac myocytes was reduced to near zero levels in a linear fashion by 1-25 microM OA. Whereas high OA concentrations reduce junctional conductance (gj) between heart cells to zero [J. M. Burt, K. D. Massey, and B. N. Minnich. Am. J. Physiol. 260 (Cell Physiol. 29): C439-C448, 1991], gj between A7r5 cells was decreased by a maximum of 45% by OA. These differences in OA sensitivity between the two cell types were not explained by differences in the rate or magnitude of OA uptake by the cells or by differences in the fraction of incorporated OA accessible to albumin washout, i.e., the plasma membrane fraction. Instead, the activity of the individual channel types exhibited different sensitivities to OA. In the presence of increasing concentrations of OA, the activities of first the 70-pS channel population [composed of connexin40 (Cx40)] and then the 108-pS channel population (composed of Cx43) were diminished, leaving predominantly the 140-pS channels (composed of Cx43) at high OA concentrations. The uncoupling effects of OA in both cell types could be reversed by washout with albumin-containing solution; however, higher concentrations of albumin and more vigorous wash conditions were required for full recovery in the A7r5 cells. In addition, albumin also reversed the effects of OA on channel activity. These data suggest that OA binds with greater affinity to the 70- vs. 108- or 140-pS channels and associated with binding is reduced channel activity.