Deborah H. Damon

Kv1.3 channels in postganglionic sympathetic neurons: Expression, function, and modulation

Kv1.3 channels are known to modulate many aspects of neuronal function. We tested the hypothesis that Kv1.3 modulates the function of postganglionic sympathetic neurons. RT-PCR, immunoblot, and immunohistochemical analyses indicated that Kv1.3 channels were expressed in these neurons. Immunohistochemical analyses indicated that Kv1.3 protein was localized to neuronal cell bodies, processes, and nerve fibers at sympathetic neurovascular junctions. Margatoxin (MgTX), a specific inhibitor of Kv1.3, was used to assess the function of the channel. Electrophysiological analyses indicated that MgTX significantly reduced outward currents [P < 0.05; n = 18 (control) and 15 (MgTX)], depolarized resting membrane potential, and decreased the latency to action potential firing [P < 0.05; n = 11 (control) and 13 (MgTX)]. The primary physiological input to postganglionic sympathetic neurons is ACh, which activates nicotinic and muscarinic ACh receptors. MgTX modulated nicotinic ACh receptor agonist-induced norepinephrine release (P < 0.05; n >or= 6), and MgTX-sensitive current was suppressed upon activation of muscarinic ACh receptors with bethanechol (P < 0.05; n = 12). These data indicate that Kv1.3 affects the function of postganglionic sympathetic neurons, which suggests that Kv1.3 influences sympathetic control of cardiovascular function. Our data also indicate that modulation of Kv1.3 is likely to affect sympathetic control of cardiovascular function.

Transforming Growth Factor-β Regulation of Endothelin Expression in Rat Vascular Cell and Organ Cultures

Transforming growth factor (TGF)-β increases the production of the vasoactive peptide endothelin (ET) in cultures of vascular endothelial cells (EC) and vascular smooth muscle cells (VSMC), but the physiologic or pathologic significance of this regulation has not been determined. The present studies test the hypothesis that when EC and VSMC are in direct contact or close proximity, ET expression is, at least in part, dependent on TGF-β. The effects of TGF-β on ET-1 mRNA (Northern analysis and reverse transcription polymerase chain reaction) and peptide (radioimmunoassay) levels were assessed in rat EC and VSMC and vascular organ cultures. TGF-β2 (1 ng/ml) increased ET-1 mRNA in VSMC and EC plus VSMC cultures and increased ET-1 peptide in EC, VSMC, and EC plus VSMC cultures. TGF-β2 also increased ET-1 mRNA and peptide in vascular organ cultures. Antibodies that neutralized the activities of TGF-β1 and TGF-β2 decreased ET-1 mRNA in EC plus VSMC cultures and in vascular organ cultures. These data indicate that when EC and VSMC are in direct contact or close proximity, TGF-β increases ET expression and active TGF-β is present and promotes ET expression. These data suggest that TGF-β is a determinant of vascular ET expression in vivo, and that TGF-β regulation of ET expression would affect cardiovascular function in health and disease.

ENDOTHELIN AND POST‐GANGLIONIC SYMPATHETIC NEURONS

1. The present report documents evidence suggesting that endothelin (ET) is a mediator and modulator of post‐ganglionic sympathetic neuronal development.

In vitro and in vivo vascular actions of basic fibroblast growth factor (bFGF) in normotensive and spontaneously hypertensive rats.

In the cardiovascular system, basic fibroblast growth factor (bFGF) is an important modulator of blood vessel growth and blood pressure and, as such, could contribute to the structural and functional changes that contribute to hypertension. This study evaluated in vitro and in vivo vascular actions of bFGF in normotensive and hypertensive rats. Basic FGF increased the expression of the messenger RNA (mRNA) encoding for the immediate early gene, egr-1, in cultured vascular smooth muscle cells (VSM) isolated from normotensive Wistar-Kyoto (WKY). Sprague-Dawley (SD), and spontaneously hypertensive rats (SHRs). Maximal increases (stimulated/control) in egr-1 mRNA were greater in SHR than in WKY and SD rat VSM (14.57 +/- 1.94 vs. 5.75 +/- 0.35 and 3.84 +/- 0.70). Basic FGF (30 ng/ml) stimulated the growth of WKY (43 +/- 8.4% of growth in 10% serum) and SD (34.6 +/- 6.5%) rat and SHR (75.8 +/- 8.8%) VSM but was most efficacious at stimulating SHR VSM. Radioligand-binding assays indicated no differences in the affinity or number of high-affinity receptors but that the binding of bFGF to low-affinity receptors was slightly but significantly greater in SHR VSM. In vivo, bFGF vasodilated cremaster arterioles in normotensive but not in hypertensive rats. These data suggest that hypertensive animals are more responsive to the growth-stimulatory actions but are less responsive to the vasodilatory actions of bFGF. This altered bFGF function could contribute to the development or maintenance or both of hypertension.

A C-terminal PDZ binding domain modulates the function and localization of Kv1.3 channels.

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms.

Eph/ephrin interactions modulate vascular sympathetic innervation

Ephs and ephrins are membrane-bound proteins that interact to modulate axon growth and neuronal function. We tested the hypothesis that eph/ephrin interactions affected the growth and function of vascular sympathetic innervation. Using RT-PCR analyses, we detected both classes of ephs (A and B) and both classes of ephrins (A and B) in sympathetic ganglia from neonatal and adult rats. Both classes of ephs (A and B) and both classes of ephrins (A and B) bound to the cell bodies and neurites of dissociated postganglionic sympathetic neurons. Messenger RNAs encoding for both classes of ephs (A and B) and both classes of ephrins (A and B) were also detected in sympathetically innervated arteries from neonatal and adult rats. These data suggest that ephrins/ephs on nerve fibers of postganglionic sympathetic neurons could interact with ephs/ephrins on cells in innervated arteries. We found that ephA4 reduced reinnervation of denervated femoral arteries. Reinnervation in the presence of ephA4-Fc (38.9±6.6%) was significantly less than that in the presence of IgG-Fc (62±10%; n=5; p<0.05; one-tailed unpaired t-test). These data indicate that eph/ephrin interactions modulated the growth of vascular sympathetic innervation. We also found that ephA4 increased basal release of norepinephrine from nerve terminals of isolated tail arteries. These data indicate that eph/ephrin interactions affect the growth and function of vascular sympathetic innervation.

Vascular endothelial growth factor protects post-ganglionic sympathetic neurones from the detrimental effects of hydrogen peroxide by increasing catalase

Aim:  Vascular production of hydrogen peroxide (H2O2) is implicated in the development and progression of vascular disease. Hydrogen peroxide also promotes neuronal degeneration, which suggests that vascular H2O2 would promote degeneration of perivascular sympathetic nerves. Vascular cells also produce vascular endothelial growth factor (VEGF), which could protect perivascular nerves from the detrimental effects of H2O2. The aim of this study was to test these hypotheses.

PC12 cells stimulate vascular smooth muscle growth

Sympathetic nerves stimulate vascular growth. The mechanisms underlying this stimulation have not been fully elucidated. PC12 cells and cultures of vascular smooth muscle were used to study sympathetic stimulation of vascular smooth muscle growth. Media conditioned by undifferentiated and differentiated PC12 cells stimulated the growth of vascular smooth muscle (446 ± 47%). Differentiated PC12 cells produced more growth-stimulatory activity (61.5 ± 9.6 per 10 6 cells) than undifferentiated PC12 cells (28.5 ± 8.8 per 10 6 cells). PC12 stimulation of vascular smooth muscle growth was not inhibited by adrenergic receptor antagonists but was reduced by an endothelin antagonist, suramin, and an antibody that neutralized the activity of platelet-derived growth factor. These data suggest that endothelin and platelet-derived growth factor, but not catecholamines, play a role in sympathetic stimulation of vascular smooth muscle growth.