The interaction between synaptic and electrotonic excitation of cells from the salivary gland of the snail Helisoma trivolvis was studied using a voltage-sensitive merocyanine dye. Linear and square photodiode matrix arrays were used to record simultaneously the response to neuronal stimulation of 15-25 separate regions of the gland . Laterally opposed acini exhibited highly synchronous electrical activity, which suggested a correspondingly high degree of electrical coupling. In the longitudinal direction, coupling appeared weaker .The onset of depolarization after neuronal stimulation was progressively delayed along the longitudinal gland axis, in agreement with the measured conduction velocity of the presynaptic nerve spike . In most instances, neuronal stimulation directly activated a regenerative gland response (action potential) at the junction between the anterior and central duct . Excitation of distal gland regions was usually mediated by electrotonic spread from active, more proximal gland regions . Occasionally, "collisions" between excitatory waves traveling in opposite directions were observed .

Diarrhea occurs in 2-50% of cases of COVID-19 (~8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2 ) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike- D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS- D614G exposure to enteroids that were pretreated for ~24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by ~8 min . The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco- 2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of ~10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

The spread of hyperpolarizing current injected into the salivary gland of the snail Helisoma trivolvis was studied with the technique of multiple site optical recording of transmembrane voltage (MSORTV). Using MSORTV it was possible to monitor simultaneously the spread of current passed by an intracellular microelectrode in up to 124 separate gland regions. Our results demonstrate the feasibility of combining conventional intracellular injection techniques with MSORTV to visualize 2-dimensional current spread in an electrical syncitium.

No abstract