Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.

Tyrosine kinases regulate intracellular calcium during α2-adrenergic contraction in rat aorta

We have demonstrated enhanced contractile sensitivity to the α2-adrenoreceptor (α2-AR) agonist UK-14304 in arteries from rats made hypertensive with chronic nitric oxide synthase (NOS) inhibition (LHR) compared with arteries from normotensive rats (NR); additionally, this contraction requires Ca2+ entry. We hypothesized that tyrosine kinases augment α2-AR contraction in LHR arteries by increasing Ca2+. The tyrosine kinase inhibitor tyrphostin 23 significantly attenuated UK-14304 contraction of denuded thoracic aortic rings from NR and LHR. However, tyrphostin 23 did not alter UK-14304 contraction in ionomycin-permeabilized aorta, which indicates that tyrosine kinases regulate intracellular Ca2+concentration. The Src family inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor AG-1478 did not alter α2-AR contraction, whereas the mitogen-activated protein kinase extracellular signal-regulated kinase kinase inhibitor PD-98059 attenuated the contraction. Contraction to CaCl2 in ionomycin-permeabilized LHR rings was greater than in NR rings. UK-14304 augmented CaCl2 contraction in ionomycin-permeabilized rings from both groups but to a greater extent in LHR aorta. Together, these data suggest that α2-AR stimulates contraction via two pathways. One, which is enhanced with NOS inhibition hypertension, activates Ca2+ sensitivity and is independent of tyrosine kinases. The other is tyrosine kinase dependent and regulates intracellular Ca2+ concentration.

Tyrosine kinase and protein kinase C regulate L-type Ca2+ current cooperatively in human atrial myocytes

The effects of tyrosine protein kinases (TK) on the L-type Ca2+ current ( I Ca) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 μM), lavendustin A (50 μM), and tyrphostin 23 (50 μM) stimulated I Ca by 132 ± 18% ( P < 0.001), 116 ± 18% ( P < 0.05), and 60 ± 6% ( P < 0.001), respectively. After I Castimulation by genistein, external application of isoproterenol (1 μM) caused an additional increase in I Ca. Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I Ca but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I Ca. The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 ± 17%, P < 0.05), decreased I Ca(−36 ± 5%, P < 0.001). Once the inhibitory effect of PMA on I Ca had stabilized, genistein strongly stimulated the current (323 ± 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I Ca. We conclude that, in human atrial myocytes, TK inhibit I Ca via a mechanism that involves PKC.

Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

We investigated the role of capacitative Ca2+ entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca2+ concentration ([Ca2+]i) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca2+]i was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca2+]i was 96 ± 4 nmol/l. PE (10 μmol/l) stimulated oscillations in [Ca2+]i, with a peak amplitude of 437 ± 22 nmol/l and a frequency of 1.01 ± 0.12/min. Thapsigargin (1 μmol/l) was used to deplete sarcoplasmic reticulum (SR) Ca2+ after extracellular Ca2+ was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca2+]i (140 ± 7% of control value) was observed when the extracellular Ca2+concentration was restored; i.e., capacitative Ca2+ entry was demonstrated. Capacitative Ca2+ entry also refilled SR Ca2+ stores. Capacitative Ca2+ entry was attenuated (32 ± 3% of control value) by 50 μmol/l of SKF-96365 (a nonselective Ca2+-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 μmol/l) and genistein (100 μmol/l) also inhibited capacitative Ca2+ entry to 63 ± 12 and 85 ± 4% of control values, respectively. SKF-96365 (30 μmol/l) attenuated both the amplitude (15 ± 7% of control value) and frequency (50 ± 21% of control value) of PE-induced Ca2+ oscillations. SKF-96365 (50 μmol/l) abolished the oscillations. Tyrphostin 23 (100 μmol/l) also inhibited the amplitude (17 ± 7% of control value) and frequency (45 ± 9% of control value) of the oscillations. Genistein (30 μmol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca2+ entry is present and capable of refilling SR Ca2+ stores in canine PASMCs and may be involved in regulating PE-induced Ca2+ oscillations. A tyrosine kinase is involved in the signal transduction pathway for α1-adrenoreceptor activation in PASMCs.

[3H]taurine andd-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases

Volume-dependent anion channels permeable for Cl− and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine andd-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at ∼40 and 1 μM, respectively. In contrast, the release ofd-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.