Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

Abstract

In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics. NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.

Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

“…In this regard, transcripts encoding all three RYR subtypes (RYR1, RYR2, and RYR3) have been detected in RNA isolated from human dermal and rat mesenteric LVs. In the latter preparation, the RYR2 and RYR3 proteins were revealed by whole-vessel mount immunostaining, but expression of the RYR1 subtype was not reported (Hasselhof et al, 2016;Jo et al, 2019). In this study, we provide initial direct evidence for the presence of RYR1 in freshly isolated populations of LMCs using a RYR1-specific antibody in flow cytometry.…”

“…Currently, DANT is available in intravenous and oral formulations (Ratto and Joyner, 2020), with a topical composition recently patented (Henry, 2014). However, DANT has not received attention as an anti-lymphedema therapeutic, largely because the contribution of RYRs to lymph muscle contraction is regarded as minimal and the identity of the RYR subtypes (RYR1, RYR2, and RYR3) expressed by LMCs is unresolved (Atchison and Johnston, 1997;Atchison et al, 1998;Zhao and van Helden, 2003;Jo et al, 2019;Stolarz et al, 2019). Accordingly, the present study also determined whether the RYR1 protein, the therapeutic target for DANT, is expressed by freshly isolated LMCs.…”

Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model

“…Additionally, while L-type calcium channel is partially responsible for differential contractile activity of mouse lymphatic vessels from various regions of the body, it controls both frequency and strength of phasic contractions in lymphatic vessel 15,60 . In the presence of ryanodine that inhibits ryanodine receptor, lymphatic vessels showed a decrease in contractile frequency and amplitude, without a change in tonic contraction 61 , suggesting critical roles of endoplasmic reticulum stored calcium in phasic contraction. Our data show that inhibition of SERCA by thapsigargin in lymphatic vessels diminished both phasic contractile frequency and amplitude without affecting the tone.…”

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow

“…However, both cardiac and some smooth muscle cells display spontaneous depolarizations, which trigger voltage-dependent Ca 21 influx through dihydropyridine-sensitive Ca v 1.2 channels to fuel rhythmic contractions, a feature also shared by lymphatic muscle cells (Atchison and Johnston, 1997;Gashev, 2002;Zawieja, 2009;Lee et al, 2014). Notably, a recent study showed amplification of transcripts encoding all three RYR isoforms from rat mesenteric collecting lymph vessels, and detected RYR2 and RYR3 proteins in the muscle layer of these vessels; studies to detect the RYR1 protein apparently were not performed (Jo et al, 2019). Interestingly, RNAseq data obtained from human dermal lymphatics also detected all three RYR isoform genes (Hasselhof et al, 2016).…”

“…In contrast, the effect of DOX on RYRs and Ca 21 signaling in lymphatic muscle cells has yet to be established. Indeed, under basal conditions, the RYRs are not regarded as contributing significantly to rhythmic contractions in isolated lymph vessels (Atchison et al, 1998;Zhao and van Helden, 2003), and the molecular identity of the functional RYRs in lymphatic muscle cells is unknown (Muthuchamy et al, 2003), although isoforms RYR2 and RYR3 recently were detected in the smooth muscle layer of rat mesenteric collecting lymph vessels (Jo et al, 2019). Regardless, we considered whether the pharmacological activation of otherwise quiescent RYRs by DOX may explain its disruption of lymphatic rhythmic contractions.…”