Biomedical subjects

Susan K Fellner

Publications and source records attributed to Susan K Fellner.

12 recordsLinked to original sources

PubMed2006

Voltage-gated Ca2+ entry and ryanodine receptor Ca2+-induced Ca2+ release in preglomerular arterioles.

We have previously shown that in afferent arterioles, angiotensin II (ANG II) involves activation of the inositol trisphosphate receptor (IP(3)R), activation of adenine diphosphoribose (ADPR) cyclase, and amplification of the initial IP(3)R-stimulated release of cytosolic Ca(2+) ([Ca(2+)](i)) from the sarcoplasmic reticulum (SR) (Fellner SK, Arendshorst WJ. Am J Physiol Renal Physiol 288: F785-F791, 2004). The response of the ryanodine receptor (RyR) to local increases in [Ca(2+)](i) is defined as calcium-induced calcium release (CICR). To investigate whether Ca(2+) entry via voltage-gated channels (VGCC) can stimulate CICR, we treated fura 2-loaded, freshly isolated afferent arterioles with KCl (40 mM; high KCl). In control arterioles, peak [Ca(2+)](i) increased by 165 +/- 10 nM. Locking the RyR in the closed position with ryanodine (100 microM) inhibited the [Ca(2+)](i) response by 59% (P < 0.01). 8-Br cADPR, a specific blocker of the ability of cyclic ADPR (cADPR) to sensitize the RyR to Ca(2+), caused a 43% inhibition. We suggest that the lower inhibition by 8-Br cADPR (P = 0.02, ryanodine vs. 8-Br cADPR) represents endogenously active ADPR cyclase. Depletion of SR Ca(2+) stores by inhibiting the SR Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin blocked the [Ca(2+)](i) responses to KCl by 51% (P not significant vs. ryanodine or 8-Br cADPR). These data suggest that about half of the increase in [Ca(2+)](i) induced by high KCl is accomplished by activation of CICR through the ability of entered Ca(2+) to expose the RyR to high local concentrations of Ca(2+) and that endogenous cADPR contributes to the process.

Animals↗

PubMed2006

Endothelin-A and -B receptors, superoxide, and Ca2+ signaling in afferent arterioles.

It is unknown if endothelin-A and -B receptors (ET(A)R and ET(B)R) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cyclic adenine diphosphate ribose (cADPR) in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura 2. Endothelin-1 (ET-1) rapidly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) by 303 nM. The superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by approximately 60%. The ET(B)R agonist sarafotoxin 6c (S6c) increased peak [Ca(2+)](i) by 117 nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca(2+)](i) peak of 225 nM. Neither nicotinamide or 8-bromo- (8-Br) cADPR nor apocynin decreased the [Ca(2+)](i) response to S6c, but inhibited the subsequent [Ca(2+)](i) response to ET-1. The ET(B)R blockers BQ-788 and A-192621 prevented the S6c [Ca(2+)](i) peak and reduced the ET-1 response by more than one-half, suggesting an ET(B)R/ET(A)R interaction. In contrast, the ET(A)R blocker BQ-123 had no effect on the S6c [Ca(2+)](i) peak and obliterated the subsequent ET-1 response. ET-1 immediately stimulated superoxide formation (measured with TEMPO-9-AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium. S6c or IRL-1620 increased superoxide by 8% of that caused by subsequent ET-1 addition. We conclude that ET(A)R activation of afferent arterioles increases the formation of superoxide that accounts for approximately 60% of subsequent Ca(2+) signaling. ET(B)R activation appears to result in only minor increases in superoxide production. Nicotinamide and 8-Br-cADPR results suggest that ET-1 (and primarily ET(A)R) causes the activation of vascular smooth muscle cell-ADPR cyclase.

ADP-ribosyl Cyclase↗

PubMed2005

Angiotensin II, reactive oxygen species, and Ca2+ signaling in afferent arterioles.

In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca(2+) ([Ca(2+)](i)) via inositol trisphosphate receptor (IP(3)R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca(2+). We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O(2)-*, which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca(2+)](i). ANG II rapidly increased [Ca(2+)](i) by 124 +/- 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca(2+)](i) response was reduced to 35 +/- 5 nM (P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca(2+)](i) response to ANG II at a concentration of 10(-4) M (99 +/- 12 nM), but 10(-3) M tempol reduced the response to 32 +/- 3 nM (P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10(-3) M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 +/- 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H(2)O(2)) was studied at physiological (10(-7) M) and higher concentrations. In the presence of H(2)O(2) (10(-7) M), neither baseline [Ca(2+)](i) nor the response to ANG II was altered (125 +/- 15 nM), whereas H(2)O(2) (10(-6) and 10(-5) M) inhibited the [Ca(2+)](i) response to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O(2)-*, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca(2+), augments the Ca(2+) response (calcium-induced calcium release) initiated by activation of the IP(3)R.

ADP-ribosyl Cyclase↗

PubMed2005

Ca2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: evidence for mobilization and entry mechanisms.

Prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.

Animals↗

PubMed2005

Endothelin-1, superoxide and adeninediphosphate ribose cyclase in shark vascular smooth muscle.

In vascular smooth muscle (VSM) of Squalus acanthias, endothelin-1 (ET-1) signals via the ET(B) receptor. In both shark and mammalian VSM, ET-1 induces a rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) via activation of the inositol trisphosphate (IP(3)) receptor (IP(3)R) and subsequent release of Ca(2+) from the sarcoplasmic reticulum (SR). IP(3)R-mediated release of SR Ca(2+) causes calcium-induced calcium release (CICR) via the ryanodine receptor (RyR), which can be sensitized by cyclic adeninediphosphate ribose (cADPR). cADPR is synthesized from NAD(+) by a membrane-bound bifunctional enzyme, ADPR cyclase. We have previously shown that the antagonists of the RyR, Ruthenium Red, high concentrations of ryanodine and 8-Br cADPR, diminish the [Ca(2+)](i) response to ET-1 in shark VSM. To investigate how ET-1 might influence the activity of the ADPR cyclase, we employed inhibitors of the cyclase. To explore the possibility that ET-1-induced production of superoxide (O(2)*-) might activate the cyclase, we used an inhibitor of NAD(P)H oxidase (NOX), DPI and a scavenger of O(2)*-, TEMPOL. Anterior mesenteric artery VSM was loaded with fura-2AM to measure [Ca(2+)](i). In Ca(2+)-free shark Ringers, ET-1 increased [Ca(2+)](i) by 104+/-8 nmol l(-1). The VSM ADPR cyclase inhibitors, nicotinamide and Zn(2+), diminished the response by 62% and 72%, respectively. Both DPI and TEMPOL reduced the response by 63%. The combination of the IP(3)R antagonists, 2-APB or TMB-8, with DPI or TEMPOL further reduced the response by 83%. We show for the first time that in shark VSM, inhibition of the ADPR cyclase reduces the [Ca(2+)](i) response to ET-1 and that superoxide may be involved in the activation of the cyclase.

ADP-ribosyl Cyclase↗

PubMed2004

Angiotensin II Ca2+ signaling in rat afferent arterioles: stimulation of cyclic ADP ribose and IP3 pathways.

ANG II induces a rise in cytosolic Ca(2+) ([Ca(2+)](i)) in vascular smooth muscle (VSM) cells via inositol trisphosphate receptor (IP(3)R) activation and release of Ca(2+) from the sarcoplasmic reticulum (SR). The Ca(2+) signal is augmented by calcium-induced calcium release (CICR) and by cyclic adeninediphosphate ribose (cADPR), which sensitizes the ryanodine-sensitive receptor (RyR) to Ca(2+) to further amplify CICR. cADPR is synthesized from beta-nicotinamide adenine dinucleotide (NAD(+)) by a membrane-bound bifunctional enzyme, ADPR cyclase. To investigate the possibility that ANG II activates the ADPR cyclase of afferent arterioles, we used inhibitors of the IP(3)R, RyR, and ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca(2+)](i). In Ca(2+)-containing buffer, ANG II increased [Ca(2+)](i) by 125 +/- 10 nM. In the presence of the IP(3)R antagonists TMB-8 and 2-APB, the peak responses to ANG II were reduced by 74 and 81%, respectively. The specific antagonist of cADPR 8-Br ADPR and a high concentration of ryanodine (100 microM) inhibited the ANG II-induced increases in [Ca(2+)](i) by 75 and 69%, respectively. Nicotinamide and Zn(2+) are known inhibitors of the VSM ADPR cyclase. Nicotinamide diminished the [Ca(2+)](i) response to ANG II by 66%. In calcium-free buffer, Zn(2+) reduced the ANG II response by 68%. Simultaneous blockade of the IP(3) and cADPR pathways diminished the [Ca(2+)](i) response to ANG II by 83%. We conclude that ANG II initiates Ca(2+) mobilization from the SR in afferent arterioles via the classic IP(3)R pathway and that ANG II may lead to activation of the ADPR cyclase to form cADPR, which, via its action on the RyR, substantially augments the Ca(2+) response.

ADP-ribosyl Cyclase↗

PubMed2004

Ionic strength and the polyvalent cation receptor of shark rectal gland and artery.

The dogfish shark Squalus acanthias regulates plasma osmolality and extracellular volume by secreting a fluid from its rectal gland which has a higher NaCl and lower urea concentration than plasma. We have previously identified the presence of a calcium-sensing receptor or polyvalent cation sensing receptor (CaSR) on vascular smooth muscle of the rectal gland artery (RGA) and rectal gland tubules (RGT). Activity of the CaSR is influenced by changes in ionic strength. This led us to speculate that the ingestion of invertebrate sea animals increased plasma ionic strength, resulting in inhibition of the receptor, relaxation of RGA, and reversal of inhibition of chloride secretion by the RGT. In contrast, ingestion of fish could diminish ionic strength and have the opposite effect. To study the effect of changes in extracellular ionic strength, shark Ringers solutions were adjusted to three different ionic strengths with NaCl, but the osmolarities were kept constant by varying the concentration of urea. High ionic strength inhibited and low ionic strength enhanced the response to increasing external Ca2+ from 2.5 to 4.7 mM in RGT. The increase in cytosolic Ca2+ ([Ca2+]i) of cells in low, normal, and high ionic strength Ringers solution was 344 +/- 60, 201 +/- 26, and 114 +/- 15 nmol/L, respectively. The [Ca2+]i responses of RGA to external Ca2+ in Ringers of three different ionic strengths were 323 +/- 43, 231 +/- 14, and 56 +/- 11 nmol/L, respectively. Activation of the CaSR by spermine was reduced by more than 50% by high ionic strength in both RGT and RGA. Whether the small changes in shark plasma ionic strength that occur after a shark ingests marine animals with lower and higher ionic strength modulates salt secretion by the rectal gland is not yet known.

Animals↗

PubMed2004

Endothelin A and B receptors of preglomerular vascular smooth muscle cells.

BACKGROUND: The endothelin (ET) receptors are subclassified into ET(A,) which are purely vasoconstrictive, and ET(B). The ET(B) receptors may cause either vasodilation by stimulating the release of nitric oxide from endothelial cells, or vasoconstriction of vascular smooth muscle cells (VSMC). The relative contribution of ET(A) and ET(B) receptors to calcium signaling and vasoconstriction in the renal microcirculation is not clear. Our goal was to study the cytosolic calcium concentration ([Ca(2+)](i)) responses of fresh rat preglomerular VSMC and afferent arterioles to agonists and antagonists of ET(A) and ET(B) receptors in rats. METHODS: Fresh VSMC and afferent arterioles were isolated using the magnetized microsphere/sieving technique, followed by gentle collagenase digestion. [Ca(2+)](i) was measured with fura-2 ratiometric fluorescence. RESULTS: Afferent arterioles and VSMC responded to ET-1 stimulation with a rapid peak increase in [Ca(2+)](i) (Delta= 287 +/- 81 and 342 +/- 55 nmol/L, respectively). The ET(B) receptor agonist IRL 1620 stimulated a rise in [Ca(2+)](i) in afferent arterioles (106 +/- 35 nmol/L); subsequent addition of ET-1 at the IRL 1620 nadir to stimulate ET(A) receptors caused a second peak that was twice as large (213 +/- 44 nmol/L). In VSMC, the ET(B) agonist peak increase was 99 +/- 12 nmol/L; addition of ET-1 then increased [Ca(2+)](i) by 294 +/- 23 nmol/L. The ET(B) inhibitor BQ-788 prevented stimulation of [Ca(2+)](i) by IRL 1620 in afferent arterioles and VSMC; subsequent stimulation of ET(A) receptors with ET-1 caused an increase in [Ca(2+)](i) (239 +/- 17 and 248 +/- 22 nmol/L). Pretreatment with the selective ET(A) inhibitor PD 156707 attenuated but did not abolish the responses to ET-1, suggesting that the residual [Ca(2+)](i) response was caused by ET(B) stimulation. CONCLUSION: These results indicate that fresh preglomerular VSMC as well as afferent arterioles have both ET(A) and ET(B) receptors, and that the rapid peak [Ca(2+)](i) responses to the ET(B) agonist IRL 1620 are less than half that of subsequent stimulation of ET(A) receptors with ET-1. The similarity of findings in isolated VSMC and afferent arterioles suggests that responses in VSMC in our arteriolar preparation overshadow any potential contribution of endothelial cells when reagents are administered abluminally.

Animals↗

PubMed2004

Endothelin B receptor Ca2+ signaling in shark vascular smooth muscle: participation of inositol trisphosphate and ryanodine receptors.

In mammals, endothelin receptors are sub-classified into ET(A) receptors (ET(A)R), which are purely constrictive in vascular smooth muscle (VSM), and ET(B)R, which may produce constriction in VSM or dilatation by stimulating the production of nitric oxide (NO) from endothelial cells. In contrast, previous studies suggested that shark VSM is stimulated exclusively by ET(B)R. The Ca(2+) signaling pathways utilized by shark VSM in response to stimulation by endothelin-1 (ET-1) have not previously been investigated. We measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded VSM of anterior mesenteric artery of Squalus acanthias and show that the ET(B)R agonists IRL 1620 and sarafotoxin S6c (SRX) increase [Ca(2+)](i) in VSM to the same extent as ET-1 and ET(B)R appears to be the only ETR subtype in sharks. To investigate the participation of the inositol trisphosphate (IP(3)) receptors (IP(3)R), we utilized two inhibitors of the mammalian IP(3)R, TMB-8 and 2-APB. In Ca(2+)-free Ringer, these agents inhibit the response to ET(B)R agonist stimulation by 71%. The ryanodine-sensitive receptor (RyR) may be activated by low concentrations of ryanodine, by abrupt local increases of [Ca(2+)](i), (calcium-induced calcium release) or by cyclic adeninediphosphate ribose (cADPR). We employed three inhibitors of activation of the RyR, Ruthenium Red, 8-Br cADPR and high concentrations of ryanodine; these agents blocked the [Ca(2+)](i) response to ET(B)R agonist stimulation by a mean of 39%. These data show for the first time that in VSM of the shark, ET(B)R activation stimulates both IP(3)R and RyR, and that cADPR is involved in RyR activation.

Analysis of Variance↗

PubMed2002

Store-operated Ca2+ entry is exaggerated in fresh preglomerular vascular smooth muscle cells of SHR.

BACKGROUND: Regulation of preglomerular vasomotor tone vessels ultimately control glomerular filtration rate, sodium reabsorption and systemic blood pressure. To gain insight into the complex renal hemodynamic factors that may result in hypertension, we studied calcium signaling pathways. METHODS: Fresh, single, preglomerular vascular smooth muscle cells (VSMC) were isolated from 5- to 6-week-old SHR and WKY utilizing a magnetized microsphere/sieving technique. Cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence. To examine store-operated calcium entry (SOC), VSMC were activated in calcium-free buffer containing nifedipine. To deplete the sarcoplasmic reticulum (SR) of Ca2+, vasopressin-1 receptor agonist [V1R; inositol trisphosphate (IP3)-mediated mobilization], ryanodine (non-IP3 induced mobilization), and cyclopiazonic acid (CPA; Ca2+-ATPase inhibition) were utilized. Addition of external calcium followed by quenching of the fura/Ca2+ signal with Mn2+ permitted assessment of divalent cation entry via SOC. RESULTS: V1R caused greater mobilization in SHR than WKY (P < 0.01) as well as greater calcium entry (P < 0.001). Ryanodine and CPA both caused SR calcium depletion that was not statistically different between strains, but absolute calcium entry through SOC was more than double in SHR following either maneuver (P < 0.001). 2-Amino-ethoxybiphenyl borane (2-APB), an inhibitor not only of IP3 receptors, but also of SOC, blocked calcium entry in the ryanodine and CPA experiments independent of IP3. As well, Gd3+, a selective inhibitor of SOC, inhibited the Ca2+ response. We also studied L-channel calcium entry stimulated by V1R. The total calcium response was greater in SHR as was the absolute inhibition by nifedipine. As a percent of the total response, participation of L-type channels sensitive to nifedipine was about 45% in both strains of rat. CONCLUSION: Utilizing three separate mechanisms to deplete the SR of Ca2+ in order to activate SOC, we show for the first time, that SOC is exaggerated in preglomerular VSMC of young SHR.

Animals↗

PubMed2002

Gordon Murray: heparin, hemodialysis and hubris.

Gordon Murray (1894-1976), a brilliant and innovative surgeon who spent the majority of his professional career at the University of Toronto, Ont., Canada, is properly credited with having performed the first successful hemodialyses in humans in North America. Neither he nor Kolff, working in the Netherlands, were aware of each other's work during the middle 1940s when wartime hampered communication. Murray's extensive investigations and experience in the use of heparin in vascular surgery laid the groundwork for the use of this anticoagulant with the artificial kidney. He first designed a coil dialyzer in which cellophane tubing was wound about a steel frame. His second-generation apparatus was a plate dialyzer. In all, he performed dialysis on 11 patients with presumed acute renal failure, 50% of whom survived. Those who died succumbed to sepsis or irreversible chronic renal failure. Not much has changed in 50 years.

Canada↗

PubMed2002

A Ca(2+)-sensing receptor modulates shark rectal gland function.

The elasmobranch Squalus acanthias controls plasma osmolality and extracellular fluid volume by secreting a hypertonic fluid from its rectal gland. Because we found a correlation between extracellular Ca(2+) concentration and changes in cytosolic Ca(2+) ([Ca(2+)](i)), we sought the possible presence of a calcium-sensing receptor in rectal gland artery and tubules. Cytosolic Ca(2+) of both tissues responded to the addition of external Ca(2+) (0.8-5.3 mmol l(-1)) in a linear fashion. Spermine, Gd(3+) and Ni(2+), known agonists of the calcium-sensing receptor, increased [Ca(2+)](i). To assess the participation of inositol triphosphate (IP(3)) generation, sarcoplasmic/endoplasmic reticulum (SR/ER) Ca(2+) depletion, and activation of store-operated Ca(2+) entry, we utilized thapsigargin and ryanodine to deplete Ca(2+) SR/ER stores and the inhibitory reagents TMB-8 and 2-APB to block IP(3) receptors. In each case, these agents inhibited the [Ca(2+)](i) response to agonist stimulation by approximately 50 %. Blockade of L-channels with nifedipine had no significant effect. Increases in ionic strength are known to inhibit the calcium-sensing receptor. We postulate that the CaSR stimulates Ca(2+)-mediated constriction of the rectal gland artery and diminishes cyclic AMP-mediated salt secretion in rectal gland tubules during non-feeding conditions. When the shark ingests sea water and fish, an increase in blood and interstitial fluid ionic strength inhibits the activity of the calcium-sensing receptor, relaxing the rectal gland artery and permitting salt secretion by the rectal gland tubules.

Animals↗